Vascular endothelial growth factor 2

ABSTRACT

Disclosed are human VEGF-2 antibodies, antibody fragments, or variants thereof. Also provided are processes for producing such antibodies. The present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder comprising administering to an animal, preferably a human, an effective amount of one or more VEGF-2 antibodies or fragments or variants thereof.

This application is a continuation of U.S. application Ser. No.11/730,696, filed Apr. 3, 2007 now U.S. Pat. No. 7,850,963, which is acontinuation of U.S. application Ser. No. 10/120,414, filed Apr. 12,2002, now U.S. Pat. No. 7,208,582 which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No.60/283,385, filed Apr. 13, 2001, and of U.S. Provisional ApplicationSer. No. 60/350,366, filed Jan. 24, 2002, all of which are hereinincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, antibodies specific forsuch polypeptides, the use of such antibodies, as well as the productionof such antibodies. The polypeptides of the present invention have beenidentified as members of the vascular endothelial growth factor family.More particularly, the polypeptides of the present invention are humanvascular endothelial growth factor 2 (VEGF-2). Antibodies of theinvention are specific for such VEGF-2 polypeptides. The invention alsorelates to inhibiting the action of such polypeptides.

The formation of new blood vessels, or angiogenesis, is essential forembryonic development, subsequent growth, and tissue repair.Angiogenesis is also an essential part of certain pathologicalconditions, such as neoplasia (i.e., tumors and gliomas). Abnormalangiogenesis is associated with other diseases such as inflammation,rheumatoid arthritis, psoriasis, and diabetic retinopathy (Folkman, J.and Klagsbrun, M., Science 235:442-447 (1987)).

Both acidic and basic fibroblast growth factor molecules are mitogensfor endothelial cells and other cell types. Angiotropin and angiogenincan induce angiogenesis, although their functions are unclear (Folkman,J., Cancer Medicine, Lea and Febiger Press, pp. 153-170 (1993)). Ahighly selective mitogen for vascular endothelial cells is vascularendothelial growth factor or VEGF (Ferrara, N. et al., Endocr. Rev.13:19-32 (1992)), which is also known as vascular permeability factor(VPF).

Vascular endothelial growth factor is a secreted angiogenic mitogenwhose target cell specificity appears to be restricted to vascularendothelial cells. The murine VEGF gene has been characterized and itsexpression pattern in embryogenesis has been analyzed. A persistentexpression of VEGF was observed in epithelial cells adjacent tofenestrated endothelium, e.g., in choroid plexus and kidney glomeruli.The data was consistent with a role of VEGF as a multifunctionalregulator of endothelial cell growth and differentiation (Breier, G. etal., Development 114:521-532 (1992)).

VEGF shares sequence homology with human platelet-derived growthfactors, PDGFa and PDGFb (Leung, D. W., et al., Science 246:1306-1309,(1989)). The extent of homology is about 21% and 23%, respectively.Eight cysteine residues contributing to disulfide-bond formation arestrictly conserved in these proteins. Although they are similar, thereare specific differences between VEGF and PDGF. While PDGF is a majorgrowth factor for connective tissue, VEGF is highly specific forendothelial cells. Alternatively spliced mRNAs have been identified forboth VEGF, PLGF, and PDGF and these different splicing products differin biological activity and in receptor-binding specificity. VEGF andPDGF function as homo-dimers or hetero-dimers and bind to receptorswhich elicit intrinsic tyrosine kinase activity following receptordimerization.

VEGF has four different forms of 121, 165, 189 and 206 amino acids dueto alternative splicing. VEGF121 and VEGF165 are soluble and are capableof promoting angiogenesis, whereas VEGF189 and VEGF-206 are bound toheparin containing proteoglycans in the cell surface. The temporal andspatial expression of VEGF has been correlated with physiologicalproliferation of the blood vessels (Gajdusek, C. M., and Carbon, S. J.,Cell Physiol. 139:570-579 (1989); McNeil, P. L., et al., J. Cell. Biol.109:811-822 (1989)). Its high affinity binding sites are localized onlyon endothelial cells in tissue sections (Jakeman, L. B., et al., Clin.Invest. 89:244-253 (1989)). The factor can be isolated from pituitarycells and several tumor cell lines, and has been implicated in somehuman gliomas (Plate, K. H., Nature 359:845-848 (1992)). Interestingly,expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cellsthe ability to form tumors in nude mice (Ferrara, N. et al., J. Clin.Invest. 91:160-170 (1993)). The inhibition of VEGF function by anti-VEGFmonoclonal antibodies was shown to inhibit tumor growth inimmune-deficient mice (Kim, K. J., Nature 362:841-844 (1993)). Further,a dominant-negative mutant of the VEGF receptor has been shown toinhibit growth of glioblastomas in mice.

Vascular permeability factor (VPF) has also been found to be responsiblefor persistent microvascular hyperpermeability to plasma proteins evenafter the cessation of injury, which is a characteristic feature ofnormal wound healing. This suggests that VPF is an important factor inwound healing. Brown, L. F. et al., J. Exp. Med. 176:1375-1379 (1992).

The expression of VEGF is high in vascularized tissues, (e.g., lung,heart, placenta and solid tumors) and correlates with angiogenesis bothtemporally and spatially. VEGF has also been shown to induceangiogenesis in vivo. Since angiogenesis is essential for the repair ofnormal tissues, especially vascular tissues, VEGF has been proposed foruse in promoting vascular tissue repair (e.g., in atherosclerosis).

U.S. Pat. No. 5,073,492, issued Dec. 17, 1991 to Chen et al., disclosesa method for synergistically enhancing endothelial cell growth in anappropriate environment which comprises adding to the environment, VEGF,effectors and serum-derived factor. Also, vascular endothelial cellgrowth factor C sub-unit DNA has been prepared by polymerase chainreaction techniques. The DNA encodes a protein that may exist as eithera heterodimer or homodimer. The protein is a mammalian vascularendothelial cell mitogen and, as such, is useful for the promotion ofvascular development and repair, as disclosed in European PatentApplication No. 92302750.2, published Sep. 30, 1992.

SUMMARY OF THE INVENTION

The polypeptides of the present invention have been putativelyidentified as a novel vascular endothelial growth factor based on aminoacid sequence homology to human VEGF.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs, andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules comprising polynucleotidesencoding full length or truncated VEGF-2 polypeptides having the aminoacid sequences shown in SEQ ID NOS:2 or 4, respectively, or the aminoacid sequences encoded by the cDNA clones deposited in bacterial hostsas ATCC Deposit Number 97149 on May 12, 1995 or ATCC Deposit Number75698 on Mar. 4, 1994.

The present invention also relates to biologically active anddiagnostically or therapeutically useful fragments, analogs, andderivatives of VEGF-2.

In accordance with still another aspect of the present invention, thereare provided processes for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said proteins and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, thereare provided processes for utilizing such polypeptides, orpolynucleotides encoding such polypeptides for therapeutic purposes, forexample, to stimulate angiogenesis, wound-healing, growth of damagedbone and tissue, and to promote vascular tissue repair. In particular,there are provided processes for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for treatment of peripheralartery disease, such as critical limb ischemia and coronary disease.

In accordance with yet another aspect of the present invention, thereare provided antibodies against such polypeptides, processes forproducing such polypeptides, and there are provided processes forutilizing such antibodies.

Using phage display technology, the present inventors have identifiedsingle chain antibody molecules (“scFvs”) that immunospecifically bindto VEGF-2, (e.g., scFvs that immunospecifically bind to full-lengthVEGF-2, scFvs that immunospecifically bind the mature form of VEGF-2polypeptide, scFvs that immunospecifically bind the pro-protein form ofVEGF-2, scFvs that immunospecifically bind the secreted form of VEGF-2and/or scFvs that immunospecifically bind to both the full-length formand the secreted form of VEGF-2. Molecules comprising, or alternativelyconsisting of, fragments or variants of these scFvs (e.g., including VHdomains, VH CDRs, VL domains, or VL CDRs having an amino acid sequenceof any one of those referred to in Table 2), that immunospecificallybind to full-length VEGF-2, the mature form of VEGF-2 polypeptide, thepro-protein form of VEGF-2, the secreted form of VEGF-2 and/or both thefull-length form and the secreted form of VEGF-2 are also encompassed bythe invention, as are nucleic acid molecules that encode these scFvs,and/or molecules.

In particular, the invention relates to scFvs comprising, oralternatively consisting of, an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 72-83 referred to in Table 2 below.Molecules comprising, or alternatively consisting of, fragments orvariants of these scFvs (e.g., including VH domains, VH CDRs, VLdomains, or VL CDRs having an amino acid sequence of any one of thosereferred to in Table 2), that immunospecifically bind to full-lengthVEGF-2, the pro-protein form of VEGF-2, the secreted form of VEGF-2and/or both the full-length form and the secreted form of VEGF-2 arealso encompassed by the invention, as are nucleic acid molecules thatencode these scFvs, and/or molecules.

The present invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that immunospecifically bind to a VEGF-2 polypeptideor polypeptide fragment or variant of a VEGF-2. In particular, theinvention encompasses antibodies (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof)that immunospecifically bind to a polypeptide or polypeptide fragment orvariant of human VEGF-2 such as those of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:18, the full length VEGF-2 polypeptide, the pro-protein form ofVEGF-2 polypeptide, the mature VEGF-2 polypeptide, or the secreted formof the VEGF-2 polypeptide.

In preferred embodiments, the invention encompasses antibodies(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof) that immunospecifically bind tofull length VEGF-2. In other preferred embodiments, the inventionencompasses antibodies (including molecules comprising, or alternativelyconsisting of, antibody fragments or variants thereof) thatimmunospecifically bind to the secreted form of VEGF-2.

The present invention relates to methods and compositions forpreventing, treating or ameliorating a disease or disorder comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that immunospecifically bind to a VEGF-2 polypeptide or afragment or variant thereof. In specific embodiments, the presentinvention relates to methods and compositions for preventing, treatingor ameliorating a disease or disorder associated with VEGF-2 function orVEGF-2 receptor (e.g., flt-4, or flk-1) function or aberrant VEGF-2 orVEGF-2 receptor (e.g., flt-4, or flk-1) expression, comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that immunospecifically bind to a VEGF-2 or a fragment orvariant thereof. In highly preferred embodiments, the present inventionrelates to antibody-based methods and compositions for preventing,treating or ameliorating tumors and tumor metastasis, particularly thoseassociated with breast, brain, colon or prostate cancers orlymphangiomas. Other diseases and disorders which can be treated,prevented and/or ameliorated with the antibodies of the inventioninclude, but are not limited to, inflammatory disorders, rheumatoidarthritis, psoriasis, diabetic retinopathy, and proliferative disorders.

The present invention also encompasses methods and compositions fordetecting, diagnosing, or prognosing diseases or disorders comprisingadministering to an animal, preferably a human, an effective amount ofone or more antibodies or fragments or variants thereof, or relatedmolecules, that immunospecifically bind to VEGF-2 or a fragment orvariant thereof. In specific embodiments, the present invention alsoencompasses methods and compositions for detecting, diagnosing, orprognosing diseases or disorders associated with VEGF-2 function orVEGF-2 receptor function or aberrant VEGF-2 or VEGF-2 receptorexpression, comprising administering to an animal, preferably a human,an effective amount of one or more antibodies or fragments or variantsthereof, or related molecules, that immunospecifically bind to VEGF-2 ora fragment or variant thereof.

In highly preferred embodiments, the present invention relates toantibody-based methods and compositions for detecting, diagnosing, orprognosing tumors and tumor metastasis, particularly those associatedwith breast, brain, colon or prostate cancers or lymphangiomas. Otherdiseases and disorders which can be detected, diagnosed, or prognosedwith the antibodies of the invention include, but are not limited to,inflammatory disorders, rheumatoid arthritis, psoriasis, diabeticretinopathy, and proliferative disorders.

Another embodiment of the present invention includes the use of theantibodies of the invention as a diagnostic tool to monitor theexpression of VEGF-2 expression in biologic samples.

The present invention also provides antibodies that bind one or moreVEGF-2 polypeptides which are coupled to a detectable label, such as anenzyme, a fluorescent label, a luminescent label, or a bioluminescentlabel. The present invention also provides antibodies that bind one ormore VEGF-2 polypeptides which are coupled to a therapeutic or cytotoxicagent. The present invention also provides antibodies that bind one ormore VEGF-2 polypeptides which are coupled to a radioactive material.

The present invention also provides antibodies that bind VEGF-2polypeptides and act as either VEGF-2 agonists or VEGF-2 antagonists.

The present invention further provides antibodies that inhibit orabolish VEGF-2 binding to its receptor (e.g., flk-1 and/or flt-4) (see,for example, Example 33). In other embodiments, the antibodies of theinvention inhibit VEGF-2 induced phosphorylation of Elk-1 (e.g., seeExample 35). In still other embodiments, the antibodies of the inventioninhibit VEGF-2 induced proliferation of vascular and or endothelial cellproliferation (e.g., see Example 34). In still other preferredembodiments, antibodies of the present invention inhibit angiogenesis(e.g., see Examples 16 or 23).

In highly preferred embodiments of the present invention, VEGF-2antibodies are used to treat, prevent or ameliorate tumors and tumormetastasis. In other highly preferred embodiments, VEGF-2 antibodies ofthe present invention are administered to an individual alone or incombination with other therapeutic compounds, especially anti-canceragents, to treat, prevent or ameliorate tumors and tumor metastasis. Instill other highly preferred embodiments, VEGF-2 antibodies of thepresent invention are administered to an individual, alone or inconjunction with other anti-cancer treatments (e.g., radiation therapyor surgery), to treat, prevent or ameliorate tumors and tumormetastasis.

The present invention also provides for a nucleic acid molecule(s),generally isolated, encoding an antibody (including molecules, such asscFvs, VH domains, or VL domains, that comprise, or alternativelyconsist of, an antibody fragment or variant thereof) of the invention.The present invention also provides a host cell transformed with anucleic acid molecule encoding an antibody (including molecules, such asscFvs, VH domains, or VL domains, that comprise, or alternativelyconsist of, an antibody fragment or variant thereof) of the inventionand progeny thereof. The present invention also provides a method forthe production of an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof) ofthe invention. The present invention further provides a method ofexpressing an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof) ofthe invention from a nucleic acid molecule. These and other aspects ofthe invention are described in further detail below.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, to prevent tumorangiogenesis and thus inhibit the growth of tumors, to treat diabeticretinopathy, inflammation, rheumatoid arthritis and psoriasis.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to nucleic acid sequences ofthe present invention.

In accordance with another aspect of the present invention, there areprovided methods of diagnosing diseases or a susceptibility to diseasesrelated to mutations in nucleic acid sequences of the present inventionand proteins encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A-E show the full length nucleotide (SEQ ID NO:1) and the deducedamino acid (SEQ ID NO:2) sequence of VEGF-2. The polypeptide comprisesapproximately 419 amino acid residues of which approximately 23represent the leader sequence. The standard one letter abbreviations foramino acids are used. Sequencing was performed using the Model 373Automated DNA Sequencer (Applied Biosystems, Inc.). Sequencing accuracyis predicted to be greater than 97%.

FIGS. 2A-D show the nucleotide (SEQ ID NO:3) and the deduced amino acid(SEQ ID NO:4) sequence of a truncated, biologically active form ofVEGF-2. The polypeptide comprises approximately 350 amino acid residuesof which approximately the first 24 amino acids represent the leadersequence.

FIGS. 3A-B are an illustration of the amino acid sequence homologybetween PDGFa (SEQ ID NO:5), PDGFb (SEQ ID NO:6), VEGF (SEQ ID NO:7),and VEGF-2 (SEQ ID NO:4). The boxed areas indicate the conservedsequences and the location of the eight conserved cysteine residues.

FIG. 4 shows, in table-form, the percent homology between PDGFa, PDGFb,VEGF, and VEGF-2.

FIG. 5 shows the presence of VEGF-2 mRNA in human breast tumor celllines.

FIG. 6 depicts the results of a Northern blot analysis of VEGF-2 inhuman adult tissues.

FIG. 7 shows a photograph of an SDS-PAGE gel after in vitrotranscription, translation and electrophoresis of the polypeptide of thepresent invention. Lane 1: ¹⁴C and rainbow M.W. marker; Lane 2: FGFcontrol; Lane 3: VEGF-2 produced by M13-reverse and forward primers;Lane 4: VEGF-2 produced by M13 reverse and VEGF-F4 primers; Lane 5:VEGF-2 produced by M13 reverse and VEGF-F5 primers.

FIGS. 8A-B depict photographs of SDS-PAGE gels. VEGF-2 polypeptide wasexpressed in a baculovirus system consisting of Sf9 cells. Protein fromthe medium and cytoplasm of cells were analyzed by SDS-PAGE undernon-reducing (FIG. 8A) and reducing (FIG. 8B) conditions.

FIG. 9 depicts a photograph of an SDS-PAGE gel. The medium from Sf9cells infected with a nucleic acid sequence of the present invention wasprecipitated. The resuspended precipitate was analyzed by SDS-PAGE andstained with Coomassie brilliant blue.

FIG. 10 depicts a photograph of an SDS-PAGE gel. VEGF-2 was purifiedfrom the medium supernatant and analyzed by SDS-PAGE in the presence orabsence of the reducing agent b-mercaptoethanol and stained by Coomassiebrilliant blue.

FIG. 11 depicts reverse phase HPLC analysis of purified VEGF-2 using aRP-300 column (0.21×3 cm, Applied Biosystems, Inc.). The column wasequilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteinseluted with a 7.5 min gradient from 0 to 60% Solvent B, composed ofacetonitrile containing 0.07% TFA. The protein elution was monitored byabsorbance at 215 nm (“red” line) and 280 nm (“blue” line). Thepercentage of Solvent B is shown by the “green” line.

FIG. 12 is a bar graph illustrating the effect of partially-purifiedVEGF-2 protein on the growth of vascular endothelial cells in comparisonto basic fibroblast growth factor.

FIG. 13 is a bar graph illustrating the effect of purified VEGF-2protein on the growth of vascular endothelial cells.

FIGS. 14A-B depicts expression of VEGF-2 mRNA in human fetal and adulttissues.

FIG. 15 depicts expression of VEGF-2 mRNA in human primary culturecells.

FIGS. 16A-B depicts transient expression of VEGF-2 protein in COS-7cells.

FIG. 17 depicts VEGF-2 stimulated proliferation of human umbilical veinendothelial cells (HUVEC).

FIG. 18 depicts VEGF-2 stimulated proliferation of dermal microvascularendothelial cells.

FIG. 19 depicts the stimulatory effect of VEGF-2 on proliferation ofmicrovascular, umbilical cord, endometrial, and bovine aorticendothelial cells.

FIGS. 20A-B depict inhibition of PDGF-induced vascular (human aortic)smooth muscle cell proliferation.

FIGS. 21A-B depicts stimulation of migration of HUVEC and bovinemicrovascular endothelial cells (BMEC) by VEGF-2.

FIG. 22 depicts stimulation of nitric oxide release of HUVEC by VEGF-2and VEGF-1.

FIG. 23 depicts inhibition of cord formation of microvascularendothelial cells (CADMEC) by VEGF-2.

FIG. 24 depicts stimulation of angiogenesis by VEGF, VEGF-2, and bFGF inthe CAM assay.

FIGS. 25A-25O depict restoration of certain parameters in the ischemiclimb by VEGF-2 protein (FIGS. 25A, 25D, 25E, 25J, and 25M) and nakedexpression plasmid (FIGS. 25B, 25F, 25G, 25K, and 25N); BP ratio (FIGS.25A-25C); Blood Flow and Flow Reserve (FIGS. 25D-25I); AngiographicScore (FIGS. 25J-25L); Capillary density (FIG. 25M-250).

FIGS. 26A-26G depict ability of VEGF-2 to affect the diastolic bloodpressure in spontaneously hypertensive rats (SHR). FIGS. 26A-26B depictthe dose-dependent decrease in diastolic blood pressure achieved withVEGF-2. FIGS. 26C-26D depict the decreased mean arterial pressure (MAP)observed with VEGF-2. FIG. 26E shows the effect of increasing doses ofVEGF-2 on the mean arterial pressure (MAP) of SHR rats. FIG. 26F showsthe effect of VEGF-2 on the diastolic pressure of SHR rats. FIG. 26Gshows the effect of VEGF-2 on the diastolic blood pressure of SHR rats.

FIG. 27 depicts inhibition of VEGF-2N and VEGF-2-induced proliferation.

FIG. 28 shows a schematic representation of the pHE4a expression vector(SEQ ID NO:16). The locations of the kanamycin resistance marker gene,the multiple cloning site linker region, the oriC sequence, and thelacIq coding sequence are indicated.

FIG. 29 shows the nucleotide sequence of the regulatory elements of thepHE4a promoter (SEQ ID NO:17). The two lac operator sequences, theShine-Delgarno sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

FIGS. 30A-F show the effect of VEGF-2 antibodies on tumor size, weight,and metastasis. FIG. 30A shows the effect of αVEGF-2 antibodies onMDA-MB-231 human breast carcinoma growth in nude mice. FIG. 30B showsthe effect of VEGF-2 antibodies on PC-3 tumor volume after 42 days ofexposure to VEGF-2 antibody. FIG. 30C shows the effect of VEGF-2antibodies on lymph node metastatic frequency. FIG. 30D shows the effectof VEGF-2 antibodies on PC-3 tumor weights after 43 days of exposure toVEGF-2 antibody. FIG. 30E shows the effect of VEGF-2 antibodies on PC-3tumor growth rate over a period just over 40 days. FIG. 30F shows theeffect of VEGF-2 antibodies on PC-3 tumor volume after 42 days ofexposure to VEGF-2 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with one aspect of the present invention, there areprovided isolated nucleic acid molecules comprising a polynucleotideencoding a VEGF-2 polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2), which was determined by sequencing a cloned cDNA.The nucleotide sequence shown in SEQ ID NO:1 was obtained by sequencinga cDNA clone, which was deposited on May 12, 1995 at the American TypeTissue Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, and given ATCC Deposit No. 97149.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules comprising a polynucleotideencoding a truncated VEGF-2 polypeptide having the deduced amino acidsequence of FIG. 2 (SEQ ID NO:4), which was determined by sequencing acloned cDNA. The nucleotide sequence shown in SEQ ID NO:3 was obtainedby sequencing a cDNA clone, which was deposited on Mar. 4, 1994 at theAmerican Type Tissue Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, and given ATCC Deposit Number 75698.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

A polynucleotide encoding a polypeptide of the present invention may beobtained from early stage human embryo (week 8 to 9) osteoclastomas,adult heart or several breast cancer cell lines. The polynucleotide ofthis invention was discovered in a cDNA library derived from early stagehuman embryo week 9. It is structurally related to the VEGF/PDGF family.It contains an open reading frame encoding a protein of about 419 aminoacid residues of which approximately the first 23 amino acid residuesare the putative leader sequence such that the mature protein comprises396 amino acids, and which protein exhibits the highest amino acidsequence homology to human vascular endothelial growth factor (30%identity), followed by PDGFa (24%) and PDGFb (22%) (See FIG. 4). It isparticularly important that all eight cysteines are conserved within allfour members of the family (see boxed areas of FIG. 3). In addition, thesignature for the PDGF/VEGF family, PXCVXXXRCXGCCN, (SEQ ID NO:8) isconserved in VEGF-2 (see FIG. 3). The homology between VEGF-2, VEGF andthe two PDGFs is at the protein sequence level. No nucleotide sequencehomology can be detected, and therefore, it would be difficult toisolate the VEGF-2 through simple approaches such as low stringencyhybridization.

The VEGF-2 polypeptide of the present invention is meant to include thefull length polypeptide and polynucleotide sequence which encodes forany leader sequences and for active fragments of the full lengthpolypeptide. Active fragments are meant to include any portions of thefull length amino acid sequence which have less than the full 419 aminoacids of the full length amino acid sequence as shown in SEQ ID NO:2,but still contain the eight cysteine residues shown conserved in FIG. 3and that still have VEGF-2 activity.

There are at least two alternatively spliced VEGF-2 mRNA sequencespresent in normal tissues. The two bands in FIG. 7, lane 5 indicate thepresence of the alternatively spliced mRNA encoding the VEGF-2polypeptide of the present invention.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 or FIG. 2, or that of the depositedclones, or may be a different coding sequence which, as a result of theredundancy or degeneracy of the genetic code, encodes the same, maturepolypeptide as the DNA of FIG. 1, FIG. 2, or the deposited cDNAs.

The polynucleotide which encodes for the mature polypeptide of FIG. 1 orFIG. 2 or for the mature polypeptides encoded by the deposited cDNAs mayinclude: only the coding sequence for the mature polypeptide; the codingsequence for the mature polypeptide and additional coding sequences suchas a leader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequences) and non-coding sequences, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs, andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 or 2, or the polypeptide encoded by the cDNA of the depositedclones. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 or 2 or the same maturepolypeptide encoded by the cDNA of the deposited clones as well asvariants of such polynucleotides which variants encode for a fragment,derivative, or analog of the polypeptides of FIG. 1 or 2, or thepolypeptide encoded by the cDNA of the deposited clones. Such nucleotidevariants include deletion variants, substitution variants, and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 or 2, or of the coding sequence of the deposited clones.As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which have a substitution, deletion or additionof one or more nucleotides, which does not substantially alter thefunction of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide which aids in expression and secretion of apolypeptide from a host cell, for example, a leader sequence whichfunctions as a secretory sequence for controlling transport of apolypeptide from the cell. The polypeptide having a leader sequence is apreprotein and may have the leader sequence cleaved by the host cell toform the mature form of the polypeptide. The polynucleotides may alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell 37:767 (1984)).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the polypeptide havingthe amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 396 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97149; (e) a nucleotidesequence encoding the mature VEGF-2 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97149;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e).

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding the polypeptide havingthe amino acid sequence in SEQ ID NO:4; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:4,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 1 to about 326 in SEQ ID NO:4; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 75698; (e) a nucleotidesequence encoding the mature VEGF-2 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 75698;or (f) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), or (e).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a VEGF-2polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the VEGF-2polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5N or 3N terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in SEQ ID NOS:1 or 3, or to the nucleotidessequence of the deposited cDNA clone(s) can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

As described in detail below, the polypeptides of the present inventioncan be used to raise polyclonal and monoclonal antibodies, which areuseful in diagnostic assays for detecting VEGF-2 protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting VEGF-2 protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” VEGF-2 protein bindingproteins which are also candidate agonist and antagonist according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Peptides that are extremely hydrophobic and those ofsix or fewer residues generally are ineffective at inducing antibodiesthat bind to the mimicked protein; longer, soluble peptides, especiallythose containing proline residues, usually are effective. Sutcliffe etal., supra, at 661. For instance, 18 of 20 peptides designed accordingto these guidelines, containing 8-39 residues covering 75% of thesequence of the influenza virus hemagglutinin HA1 polypeptide chain,induced antibodies that reacted with the HA1 protein or intact virus;and 12/12 peptides from the MuLV polymerase and 18/18 from the rabiesglycoprotein induced antibodies that precipitated the respectiveproteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. Sutcliffe et al., supra, at663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptide and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, for instance, Wilsonet al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies ofthe invention also are useful for purification of the mimicked protein,for instance, by adsorption chromatography using methods well known inthe art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence of apolypeptide of the invention, containing about 30, 40, 50, 60, 70, 80,90, 100, or 150 amino acids, or any length up to and including theentire amino acid sequence of a polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); and sequencescontaining proline residues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate VEGF-2-specific antibodies include the following: apolypeptide comprising amino acid residues from about leu-37 to aboutglu-45 in SEQ ID NO:2, from about Tyr-58 to about Gly-66 in SEQ ID NO:2,from about Gln-73 to about Glu-81 in SEQ ID NO:2, from about Asp-100 toabout Cys-108 in SEQ ID NO:2, from about Gly-140 to about Leu-148 in SEQID NO:2, from about Pro-168 to about Val-176 in SEQ ID NO:2, from aboutHis-183 to about Lys-191 in SEQ ID NO:2, from about Ile-201 to aboutThr-209 in SEQ ID NO:2, from about Ala-216 to about Tyr-224 in SEQ IDNO:2, from about Asp-244 to about His-254 in SEQ ID NO:2, from aboutGly-258 to about Glu-266 in SEQ ID NO:2, from about Cys-272 to aboutSer-280 in SEQ ID NO:2, from about Pro-283 to about Ser-291 in SEQ IDNO:2, from about Cys-296 to about Gln-304 in SEQ ID NO:2, from aboutAla-307 to about Cys-316 in SEQ ID NO:2, from about Val-319 to aboutCys-335 in SEQ ID NO:2, from about Cys-339 to about Leu-347 in SEQ IDNO:2, from about Cys-360 to about Glu-373 in SEQ ID NO:2, from aboutTyr-378 to about Val-386 in SEQ ID NO:2, and from about Ser-388 to aboutSer-396 in SEQ ID NO:2. These polypeptide fragments have been determinedto bear antigenic epitopes of the VEGF-2 protein by the analysis of theJameson-Wolf antigenic index.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, a short epitope-bearing amino acid sequence maybe fused to a larger polypeptide that acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple method for synthesis of large numbers ofpeptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 mg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a Amimotope) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

As one of skill in the art will appreciate, VEGF-2 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)).

In accordance with the present invention, novel variants of VEGF-2 arealso described. These can be produced by deleting or substituting one ormore amino acids of VEGF-2. Natural mutations are called allelicvariations. Allelic variations can be silent (no change in the encodedpolypeptide) or may have altered amino acid sequence.

In order to attempt to improve or alter the characteristics of nativeVEGF-2, protein engineering may be employed. Recombinant DNA technologyknown to those skilled in the art can be used to create novelpolypeptides. Muteins and deletions can show, e.g., enhanced activity orincreased stability. In addition, they could be purified in higher yieldand show better solubility at least under certain purification andstorage conditions. Set forth below are examples of mutations that canbe constructed.

Amino Terminal and Carboxy Terminal Deletions

Furthermore, VEGF-2 appears to be proteolytically cleaved uponexpression resulting in polypeptide fragments of the following sizeswhen run on a SDS-PAGE gel (sizes are approximate) (See, FIGS. 6-8, forexample): 80, 59, 45, 43, 41, 40, 39, 38, 37, 36, 31, 29, 21, and 15kDa. These polypeptide fragments are the result of proteolytic cleavageat both the N-terminal and C-terminal portions of the protein. Theseproteolytically generated fragments appears to have activity,particularly the 21 kDa fragment.

In addition, protein engineering may be employed in order to improve oralter one or more characteristics of native VEGF-2. The deletion ofcarboxyterminal amino acids can enhance the activity of proteins. Oneexample is interferon gamma that shows up to ten times higher activityby deleting ten amino acid residues from the carboxy terminus of theprotein (Döbeli et al., J of Biotechnology 7:199-216 (1988)). Thus, oneaspect of the invention is to provide polypeptide analogs of VEGF-2 andnucleotide sequences encoding such analogs that exhibit enhancedstability (e.g., when exposed to typical pH, thermal conditions or otherstorage conditions) relative to the native VEGF-2 polypeptide.

Particularly preferred VEGF-2 polypeptides are shown below (numberingstarts with the first amino acid in the protein (Met) (FIG. 1 (SEQ IDNO: 18)): Ala (residue 25) to Ser (residue 419); Pro (26) to Ser (419);Ala (27) to Ser (419); Ala (28) to Ser (419); Ala (29) to Ser (419); Ala(30) to Ser (419); Ala (31) to Ser (419); Phe (32) to Ser (419); Glu(33) to Ser (419); Ser (34) to Ser (419); Gly (35) to Ser (419); Leu(36) to Ser (419); Asp (37) to Ser (419); Leu (38) to (Ser (419); Ser(39) to Ser (419); Asp (40) to Ser (419); Ala (41) to Ser (419); Glu(42) to Ser (419); Pro (43) to Ser (419); Asp (44) to Ser (419); Ala(45) to Ser (419); Gly (46) to Ser (419); Glu (47) to Ser (419); Ala(48) to Ser (419); Thr (49) to Ser (419); Ala (50) to Ser (419); Tyr(51) to Ser (419); Ser (53) to Ser (419); Asp (55) Ser (419); Val (63)to Ser (419); Val (66) to Ser (419); Met (1), Glu (24), or Ala (25) toMet (418); Met (1), Glu (24), or Ala (25) to Gln (417); Met (1), Glu(24), or Ala (25) to Pro (416); Met (1), Glu (24), or Ala (25) to Arg(415); Met (1), Glu (24), or Ala (25) to Gln (414); Met (1), Glu (24),or Ala (25) to Trp (413); Met (1), Glu (24), or Ala (25) to Tyr (412);Met (1), Glu (24), or Ala (25) to Ser (411); Met (1), Glu (24), or Ala(25) to Pro (410); Met (1), Glu (24), or Ala (25) to Val (409); Met (1),Glu (24), or Ala (25) to Cys (408); Met (1), Glu (24), or Ala (25) toArg (407); Met (1), Glu (24), or Ala (25) to Cys (406); Met (1), Glu(24), or Ala (25) to Val (405); Met (1), Glu (24), or Ala (25) to Glu(404); Met (1), Glu (24), or Ala (25) to Glu (403); Met (1), Glu (24),or Ala (25) to Ser (402); Met (1), Glu (24), or Ala (25) to Gly (398);Met (1), Glu (24), or Ala (25) to Pro (397); Met (1), Glu (24), or Ala(25) to Lys (393); Met (1), Glu (24), or Ala (25) to Met (263); Met (1),Glu (24), or Ala (25) to Asp (311); Met (1), Glu (24), or Ala (25) toPro (366); Met (1) to Ser (419); Met (1) to Ser (228); Glu (47) to Ser(419); Ala (111) to Lys (214); Ala (112) to Lys (214); His (113) to Lys(214); Tyr (114) to Lys (214); Asn (115) to Lys (214); Thr (116) to Lys(214); Thr (103) to Leu (215); Glu (104) to Leu (215); Glu (105) to Leu(215); Thr (106) to Leu (215); Ile (107) to Leu (215); Lys (108) to Leu(215); Phe (109) to Leu (215); Ala (10) to Leu (215); Ala (111) to Leu(215); Ala (112) to Leu (215); His (113) to Leu (215); Tyr (114) to Leu(215); Asn (115) to Leu (215); Thr (116) to Leu (215); Thr (103) to Ser(228); Glu (104) to Ser (228); Glu (105) to Ser (228); Thr (106) to Ser(228); Ile (107) to Ser (228); Lys (108) to Ser (228); Phe (109) to Ser(228); Ala (110) to Ser (228); Ala (111) to Ser (228); Ala (112) to Ser(228); His (113) to Ser (228); Tyr (114) to Ser (228); Asn (115) to Ser(228); Thr (116) to Ser (228); Thr (103) to Leu (229); Glu (104) to Leu(229); Thr (103) to Arg (227); Glu (104) to Arg (227); Glu (105) to Arg(227); Thr (106) to Arg (227); Ile (107) to Arg (227); Lys (108) to Arg(227); Phe (109) to Arg (227); Ala (110) to Arg (227); Ala (111) to Arg(227); Ala (112) to Arg (227); His (113) to Arg (227); Tyr (114) to Arg(227); Asn (115) to Arg (227); Thr (116) to Mg (227); Thr (103) to Ser(213); Glu (104) to Ser (213); Glu (105) to Ser (213); Thr (106) to Ser(213); Ile (107) to Ser (213); Lys (108) to Ser (213); Phe (109) to Ser(213); Ala (110) to Ser (213); Ala (111) to Ser (213); Ala (112) to Ser(213); His (113) to Ser (213); Tyr (114) to Ser (213); Asn (115) to Ser(213); Thr (116) to Ser (213); Thr (103) to Lys (214); Glu (104) to Lys(214); Glu (105) to Lys (214); Thr (106) to Lys (214); Ile (107) to Lys(214); Lys (108) to Lys (214); Phe (109) to Lys (214); Ala (110) to Lys(214); Glu (105) to Leu (229); Thr (106) to Leu (229); Ile (107) to Leu(229); Lys (108) to Leu (229); Phe (109) to Leu (229); Ala (110) to Leu(229); Ala (111) to Leu (229); Ala (112) to Leu (229); His (113) to Leu(229); Tyr (114) to Leu (229); Asn (115) to Leu (229); Thr (116) to Leu(229).

Preferred embodiments include the following deletion mutants: Thr(103)-Arg (227); Glu (104)-Arg (227); Ala (112)-Arg (227); Thr (103)-Ser(213); Glu (104)-Ser (213); Thr (103)-Leu (215); Glu (47)-Ser (419); Met(1), Glu (24), or Ala (25)-Met (263); Met (1), Glu (24), or Ala (25)-Asp(311); Met (1), Glu (24), or Ala (25)-Pro (366); Met (1)-Ser (419); andMet (1)-Ser (228) of (FIG. 1 (SEQ ID NO: 18)).

Also included by the present invention are deletion mutants having aminoacids deleted from both the N-terminus and the C-terminus. Such mutantsinclude all combinations of the N-terminal deletion mutants andC-terminal deletion mutants described above. Those combinations can bemade using recombinant techniques known to those skilled in the art.

Particularly, N-terminal deletions of the VEGF-2 polypeptide can bedescribed by the general formula m-396, where m is an integer from −23to 388, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. Preferably, N-terminal deletions retain theconserved boxed area of FIG. 3 (PXCVXXXRCXGCCN) (SEQ ID NO:8).N-terminal deletions of the polypeptide of the invention shown as SEQ IDNO:2 include polypeptides comprising the amino acid sequence ofresidues: E-1 to S-396; A-2 to S-396; P-3 to S-396; A-4 to S-396; A-5 toS-396; A-6 to S-396; A-7 to S-396; A-8 to S-396; F-9 to S-396; E-10 toS-396; S-11 to S-396; G-12 to S-396; L-13 to S-396; D-14 to S-396; L-15to S-396; S-16 to S-396; D-17 to S-396; A-18 to S-396; E-19 to S-396;P-20 to S-396; D-21 to S-396; A-22 to S-396; G-23 to S-396; E-24 toS-396; A-25 to S-396; T-26 to S-396; A-27 to S-396; Y-28 to S-396; A-29to S-396; S-30 to S-396; K-31 to S-396; D-32 to S-396; L-33 to S-396;E-34 to S-396; E-35 to S-396; Q-36 to S-396; L-37 to S-396; R-38 toS-396; S-39 to S-396; V-40 to S-396; S-41 to S-396; S-42 to S-396; V-43to S-396; D-44 to S-396; E-45 to S-396; L-46 to S-396; M-47 to S-396;T-48 to S-396; V-49 to S-396; L-50 to S-396; Y-51 to S-396; P-52 toS-396; E-53 to S-396; Y-54 to S-396; W-55 to S-396; K-56 to S-396; M-57to S-396; Y-58 to S-396; K-59 to S-396; C-60 to S-396; Q-61 to S-396;L-62 to S-396; R-63 to S-396; K-64 to S-396; G-65 to S-396; G-66 toS-396; W-67 to S-396; Q-68 to S-396; H-69 to S-396; N-70 to S-396; R-71to S-396; E-72 to S-396; Q-73 to S-396; A-74 to S-396; N-75 to S-396;L-76 to S-396; N-77 to S-396; S-78 to S-396; R-79 to S-396; T-80 toS-396; E-81 to S-396; E-82 to S-396; T-83 to S-396; I-84 to S-396; K-85to S-396; F-86 to S-396; A-87 to S-396; A-88 to S-396; A-89 to S-396;H-90 to S-396; Y-91 to S-396; N-92 to S-396; T-93 to S-396; E-94 toS-396; I-95 to S-396; L-96 to S-396; K-97 to S-396; S-98 to S-396; I-99to S-396; D-100 to S-396; N-101 to S-396; E-102 to S-396; W-103 toS-396; R-104 to S-396; K-105 to S-396; T-106 to S-396; Q-107 to S-396;C-108 to S-396; M-109 to S-396; P-110 to S-396; R-111 to S-396; E-112 toS-396; V-113 to S-396; C-114 to S-396; I-115 to S-396; D-116 to S-396;V-117 to S-396; G-118 to S-396; K-119 to S-396; E-120 to S-396; F-121 toS-396; G-122 to S-396; V-123 to S-396; A-124 to S-396; T-125 to S-396;N-126 to S-396; T-127 to S-396; F-128 to S-396; F-129 to S-396; K-130 toS-396; P-131 to S-396; P-132 to S-396; C-133 to S-396; V-134 to S-396;S-135 to S-396; V-136 to S-396; Y-137 to S-396; R-138 to S-396; C-139 toS-396; G-140 to S-396; G-141 to S-396; C-142 to S-396; C-143 to S-396;N-144 to S-396; S-145 to S-396; E-146 to S-396; G-147 to S-396; L-148 toS-396; Q-149 to S-396; C-150 to S-396; M-151 to S-396; N-152 to S-396;T-153 to S-396; S-154 to S-396; T-155 to S-396; S-156 to S-396; Y-157 toS-396; L-158 to S-396; S-159 to S-396; K-160 to S-396; T-161 to S-396;L-162 to S-396; F-163 to S-396; E-164 to S-396; I-165 to S-396; T-166 toS-396; V-167 to S-396; P-168 to S-396; L-169 to S-396; S-170 to S-396;Q-171 to S-396; G-172 to S-396; P-173 to S-396; K-174 to S-396; P-175 toS-396; V-176 to S-396; T-177 to S-396; I-178 to S-396; S-179 to S-396;F-180 to S-396; A-181 to S-396; N-182 to S-396; H-183 to S-396; T-184 toS-396; S-185 to S-396; C-186 to S-396; R-187 to S-396; C-188 to S-396;M-189 to S-396; S-190 to S-396; K-191 to S-396; L-192 to S-396; D-193 toS-396; V-194 to S-396; Y-195 to S-396; R-196 to S-396; Q-197 to S-396;V-198 to S-396; H-199 to S-396; S-200 to S-396; I-201 to S-396; I-202 toS-396; R-203 to S-396; R-204 to S-396; S-205 to S-396; L-206 to S-396;P-207 to S-396; A-208 to S-396; T-209 to S-396; L-210 to S-396; P-211 toS-396; Q-212 to S-396; C-213 to S-396; Q-214 to S-396; A-215 to S-396;A-216 to S-396; N-217 to S-396; K-218 to S-396; T-219 to S-396; C-220 toS-396; P-221 to S-396; T-222 to S-396; N-223 to S-396; Y-224 to S-396;M-225 to S-396; W-226 to S-396; N-227 to S-396; N-228 to S-396; H-229 toS-396; I-230 to S-396; C-231 to S-396; R-232 to S-396; C-233 to S-396;L-234 to S-396; A-235 to S-396; Q-236 to S-396; E-237 to S-396; D-238 toS-396; F-239 to S-396; M-240 to S-396; F-241 to S-396; S-242 to S-396;S-243 to S-396; D-244 to S-396; A-245 to S-396; G-246 to S-396; D-247 toS-396; D-248 to S-396; S-249 to S-396; T-250 to S-396; D-251 to S-396;G-252 to S-396; F-253 to S-396; H-254 to S-396; D-255 to S-396; I-256 toS-396; C-257 to S-396; G-258 to S-396; P-259 to S-396; N-260 to S-396;K-261 to S-396; E-262 to S-396; L-263 to S-396; D-264 to S-396; E-265 toS-396; E-266 to S-396; T-267 to S-396; C-268 to S-396; Q-269 to S-396;C-270 to S-396; V-271 to S-396; C-272 to S-396; R-273 to S-396; A-274 toS-396; G-275 to S-396; L-276 to S-396; R-277 to S-396; P-278 to S-396;A-279 to S-396; S-280 to S-396; C-281 to S-396; G-282 to S-396; P-283 toS-396; H-284 to S-396; K-285 to S-396; E-286 to S-396; L-287 to S-396;D-288 to S-396; R-289 to S-396; N-290 to S-396; S-291 to S-396; C-292 toS-396; Q-293 to S-396; C-294 to S-396; V-295 to S-396; C-296 to S-396;K-297 to S-396; N-298 to S-396; K-299 to S-396; L-300 to S-396; F-301 toS-396; P-302 to S-396; S-303 to S-396; Q-304 to S-396; C-305 to S-396;G-306 to S-396; A-307 to S-396; N-308 to S-396; R-309 to S-396; E-310 toS-396; F-311 to S-396; D-312 to S-396; E-313 to S-396; N-314 to S-396;T-315 to S-396; C-316 to S-396; Q-317 to S-396; C-318 to S-396; V-319 toS-396; C-320 to S-396; K-321 to S-396; R-322 to S-396; T-323 to S-396;C-324 to S-396; P-325 to S-396; R-326 to S-396; N-327 to S-396; Q-328 toS-396; P-329 to S-396; L-330 to S-396; N-331 to S-396; P-332 to S-396;G-333 to S-396; K-334 to S-396; C-335 to S-396; A-336 to S-396; C-337 toS-396; E-338 to S-396; C-339 to S-396; T-340 to S-396; E-341 to S-396;S-342 to S-396; P-343 to S-396; Q-344 to S-396; K-345 to S-396; C-346 toS-396; L-347 to S-396; L-348 to S-396; K-349 to S-396; G-350 to S-396;K-351 to S-396; K-352 to S-396; F-353 to S-396; H-354 to S-396; H-355 toS-396; Q-356 to S-396; T-357 to S-396; C-358 to S-396; S-359 to S-396;C-360 to S-396; Y-361 to S-396; R-362 to S-396; R-363 to S-396; P-364 toS-396; C-365 to S-396; T-366 to S-396; N-367 to S-396; R-368 to S-396;Q-369 to S-396; K-370 to S-396; A-371 to S-396; C-372 to S-396; E-373 toS-396; P-374 to S-396; G-375 to S-396; F-376 to S-396; S-377 to S-396;Y-378 to S-396; S-379 to S-396; E-380 to S-396; E-381 to S-396; V-382 toS-396; C-383 to S-396; R-384 to S-396; C-385 to S-396; V-386 to S-396;P-387 to S-396; S-388 to S-396; Y-389 to S-396; W-390 to S-396; Q-391 toS-396 of SEQ ID NO:2. One preferred embodiment comprises amino acidsS-205 to S-396 of SEQ ID NO:2. Also preferred are polynucleotidesencoding these polypeptides.

Moreover, C-terminal deletions of the VEGF-2 polypeptide can also bedescribed by the general formula −23-n, where n is an integer from −15to 395 where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. Preferably, C-terminal deletions retain theconserved boxed area of FIG. 3 (PXCVXXXRCXGCCN) (SEQ ID NO:8).C-terminal deletions of the polypeptide of the invention shown as SEQ IDNO:2 include polypeptides comprising the amino acid sequence ofresidues: E-1 to M-395; E-1 to Q-394; E-1 to P-393; E-1 to R-392; E-1 toQ-391; E-1 to W-390; E-1 to Y-389; E-1 to S-388; E-1 to P-387; E-1 toV-386; E-1 to C-385; E-1 to R-384; E-1 to C-383; E-1 to V-382; E-1 toE-381; E-1 to E-380; E-1 to S-379; E-1 to Y-378; E-1 to S-377; E-1 toF-376; E-1 to G-375; E-1 to P-374; E-1 to E-373; E-1 to C-372; E-1 toA-371; E-1 to K-370; E-1 to Q-369; E-1 to R-368; E-1 to N-367; E-1 toT-366; E-1 to C-365; E-1 to P-364; E-1 to R-363; E-1 to R-362; E-1 toY-361; E-1 to C-360; E-1 to S-359; E-1 to C-358; E-1 to T-357; E-1 toQ-356; E-1 to H-355; E-1 to H-354; E-1 to F-353; E-1 to K-352; E-1 toK-351; E-1 to G-350; E-1 to K-349; E-1 to L-348; E-1 to L-347; E-1 toC-346; E-1 to K-345; E-1 to Q-344; E-1 to P-343; E-1 to S-342; E-1 toE-341; E-1 to T-340; E-1 to C-339; E-1 to E-338; E-1 to C-337; E-1 toA-336; E-1 to C-335; E-1 to K-334; E-1 to G-333; E-1 to P-332; E-1 toN-331; E-1 to L-330; E-1 to P-329; E-1 to Q-328; E-1 to N-327; E-1 toR-326; E-1 to P-325; E-1 to C-324; E-1 to T-323; E-1 to R-322; E-1 toK-321; E-1 to C-320; E-1 to V-319; E-1 to C-318; E-1 to Q-317; E-1 toC-316; E-1 to T-315; E-1 to N-314; E-1 to E-313; E-1 to D-312; E-1 toF-311; E-1 to E-310; E-1 to R-309; E-1 to N-308; E-1 to A-307; E-1 toG-306; E-1 to C-305; E-1 to Q-304; E-1 to S-303; E-1 to P-302; E-1 toF-301; E-1 to L-300; E-1 to K-299; E-1 to N-298; E-1 to K-297; E-1 toC-296; E-1 to V-295; E-1 to C-294; E-1 to Q-293; E-1 to C-292; E-1 toS-291; E-1 to N-290; E-1 to R-289; E-1 to D-288; E-1 to L-287; E-1 toE-286; E-1 to K-285; E-1 to H-284; E-1 to P-283; E-1 to G-282; E-1 toC-281; E-1 to S-280; E-1 to A-279; E-1 to P-278; E-1 to R-277; E-1 toL-276; E-1 to G-275; E-1 to A-274; E-1 to R-273; E-1 to C-272; E-1 toV-271; E-1 to C-270; E-1 to Q-269; E-1 to C-268; E-1 to T-267; E-1 toE-266; E-1 to E-265; E-1 to D-264; E-1 to L-263; E-1 to E-262; E-1 toK-261; E-1 to N-260; E-1 to P-259; E-1 to G-258; E-1 to C-257; E-1 toI-256; E-1 to D-255; E-1 to H-254; E-1 to F-253; E-1 to G-252; E-1 toD-251; E-1 to T-250; E-1 to S-249; E-1 to D-248; E-1 to D-247; E-1 toG-246; E-1 to A-245; E-1 to D-244; E-1 to S-243; E-1 to S-242; E-1 toF-241; E-1 to M-240; E-1 to F-239; E-1 to D-238; E-1 to E-237; E-1 toQ-236; E-1 to A-235; E-1 to L-234; E-1 to C-233; E-1 to R-232; E-1 toC-231; E-1 to I-230; E-1 to H-229; E-1 to N-228; E-1 to N-227; E-1 toW-226; E-1 to M-225; E-1 to Y-224; E-1 to N-223; E-1 to T-222; E-1 toP-221; E-1 to C-220; E-1 to T-219; E-1 to K-218; E-1 to N-217; E-1 toA-216; E-1 to A-215; E-1 to Q-214; E-1 to C-213; E-1 to Q-212; E-1 toP-211; E-1 to L-210; E-1 to T-209; E-1 to A-208; E-1 to P-207; E-1 toL-206; E-1 to S-205; E-1 to R-204; E-1 to R-203; E-1 to I-202; E-1 toI-201; E-1 to S-200; E-1 to H-199; E-1 to V-198; E-1 to Q-197; E-1 toR-196; E-1 to Y-195; E-1 to V-194; E-1 to D-193; E-1 to L-192; E-1 toK-191; E-1 to S-190; E-1 to M-189; E-1 to C-188; E-1 to R-187; E-1 toC-186; E-1 to S-185; E-1 to T-184; E-1 to H-183; E-1 to N-182; E-1 toA-181; E-1 to F-180; E-1 to S-179; E-1 to I-178; E-1 to T-177; E-1 toV-176; E-1 to P-175; E-1 to K-174; E-1 to P-173; E-1 to G-172; E-1 toQ-171; E-1 to S-170; E-1 to L-169; E-1 to P-168; E-1 to V-167; E-1 toT-166; E-1 to I-165; E-1 to E-164; E-1 to F-163; E-1 to L-162; E-1 toT-161; E-1 to K-160; E-1 to S-159; E-1 to L-158; E-1 to Y-157; E-1 toS-156; E-1 to T-155; E-1 to S-154; E-1 to T-153; E-1 to N-152; E-1 toM-151; E-1 to C-150; E-1 to Q-149; E-1 to L-148; E-1 to G-147; E-1 toE-146; E-1 to S-145; E-1 to N-144; E-1 to C-143; E-1 to C-142; E-1 toG-141; E-1 to G-140; E-1 to C-139; E-1 to R-138; E-1 to Y-137; E-1 toV-136; E-1 to S-135; E-1 to V-134; E-1 to C-133; E-1 to P-132; E-1 toP-131; E-1 to K-130; E-1 to F-129; E-1 to F-128; E-1 to T-127; E-1 toN-126; E-1 to T-125; E-1 to A-124; E-1 to V-123; E-1 to G-122; E-1 toF-121; E-1 to E-120; E-1 to K-119; E-1 to G-118; E-1 to V-117; E-1 toD-116; E-1 to I-115; E-1 to C-114; E-1 to V-113; E-1 to E-112; E-1 toR-111; E-1 to P-110; E-1 to M-109; E-1 to C-108; E-1 to Q-107; E-1 toT-106; E-1 to K-105; E-1 to R-104; E-1 to W-103; E-1 to E-102; E-1 toN-101; E-1 to D-100; E-1 to I-99; E-1 to S-98; E-1 to K-97; E-1 to L-96;E-1 to I-95; E-1 to E-94; E-1 to T-93; E-1 to N-92; E-1 to Y-91; E-1 toH-90; E-1 to A-89; E-1 to A-88; E-1 to A-87; E-1 to F-86; E-1 to K-85;E-1 to I-84; E-1 to T-83; E-1 to E-82; E-1 to E-81; E-1 to T-80; E-1 toR-79; E-1 to S-78; E-1 to N-77; E-1 to L-76; E-1 to N-75; E-1 to A-74;E-1 to Q-73; E-1 to E-72; E-1 to R-71; E-1 to N-70; E-1 to H-69; E-1 toQ-68; E-1 to W-67; E-1 to G-66; E-1 to G-65; E-1 to K-64; E-1 to R-63;E-1 to L-62; E-1 to Q-61; E-1 to C-60; E-1 to K-59; E-1 to Y-58; E-1 toM-57; E-1 to K-56; E-1 to W-55; E-1 to Y-54; E-1 to E-53; E-1 to P-52;E-1 to Y-51; E-1 to L-50; E-1 to V-49; E-1 to T-48; E-1 to M-47; E-1 toL-46; E-1 to E-45; E-1 to D-44; E-1 to V-43; E-1 to S-42; E-1 to S-41;E-1 to V-40; E-1 to S-39; E-1 to R-38; E-1 to L-37; E-1 to Q-36; E-1 toE-35; E-1 to E-34; E-1 to L-33; E-1 to D-32; E-1 to K-31; E-1 to S-30;E-1 to A-29; E-1 to Y-28; E-1 to A-27; E-1 to T-26; E-1 to A-25; E-1 toE-24; E-1 to G-23; E-1 to A-22; E-1 to D-21; E-1 to P-20; E-1 to E-19;E-1 to A-18; E-1 to D-17; E-1 to S-16; E-1 to L-15; E-1 to D-14; E-1 toL-13; E-1 to G-12; E-1 to S-11; E-1 to E-10; E-1 to F-9; E-1 to A-8; E-1to A-7 of SEQ ID NO:2. Also preferred are polynucleotides encoding thesepolypeptides.

Moreover, the invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini, whichmay be described generally as having residues m-n of SEQ ID NO:2, wheren and m are integers as described above.

Likewise, also preferred are C-terminal deletions of the VEGF-2polypeptide of the invention shown as SEQ ID NO:2 which includepolypeptides comprising the amino acid sequence of residues: F-9 toM-395; F-9 to Q-394; F-9 to P-393; F-9 to R-392; F-9 to Q-391; F-9 toW-390; F-9 to Y-389; F-9 to S-388; F-9 to P-387; F-9 to V-386; F-9 toC-385; F-9 to R-384; F-9 to C-383; F-9 to V-382; F-9 to E-381; F-9 toE-380; F-9 to S-379; F-9 to Y-378; F-9 to S-377; F-9 to F-376; F-9 toG-375; F-9 to P-374; F-9 to E-373; F-9 to C-372; F-9 to A-371; F-9 toK-370; F-9 to Q-369; F-9 to R-368; F-9 to N-367; F-9 to T-366; F-9 toC-365; F-9 to P-364; F-9 to R-363; F-9 to R-362; F-9 to Y-361; F-9 toC-360; F-9 to S-359; F-9 to C-358; F-9 to T-357; F-9 to Q-356; F-9 toH-355; F-9 to H-354; F-9 to F-353; F-9 to K-352; F-9 to K-351; F-9 toG-350; F-9 to K-349; F-9 to L-348; F-9 to L-347; F-9 to C-346; F-9 toK-345; F-9 to Q-344; F-9 to P-343; F-9 to S-342; F-9 to E-341; F-9 toT-340; F-9 to C-339; F-9 to E-338; F-9 to C-337; F-9 to A-336; F-9 toC-335; F-9 to K-334; F-9 to G-333; F-9 to P-332; F-9 to N-331; F-9 toL-330; F-9 to P-329; F-9 to Q-328; F-9 to N-327; F-9 to R-326; F-9 toP-325; F-9 to C-324; F-9 to T-323; F-9 to R-322; F-9 to K-321; F-9 toC-320; F-9 to V-319; F-9 to C-318; F-9 to Q-317; F-9 to C-316; F-9 toT-315; F-9 to N-314; F-9 to E-313; F-9 to D-312; F-9 to F-311; F-9 toE-310; F-9 to R-309; F-9 to N-308; F-9 to A-307; F-9 to G-306; F-9 toC-305; F-9 to Q-304; F-9 to S-303; F-9 to P-302; F-9 to F-301; F-9 toL-300; F-9 to K-299; F-9 to N-298; F-9 to K-297; F-9 to C-296; F-9 toV-295; F-9 to C-294; F-9 to Q-293; F-9 to C-292; F-9 to S-291; F-9 toN-290; F-9 to R-289; F-9 to D-288; F-9 to L-287; F-9 to E-286; F-9 toK-285; F-9 to H-284; F-9 to P-283; F-9 to G-282; F-9 to C-281; F-9 toS-280; F-9 to A-279; F-9 to P-278; F-9 to R-277; F-9 to L-276; F-9 toG-275; F-9 to A-274; F-9 to R-273; F-9 to C-272; F-9 to V-271; F-9 toC-270; F-9 to Q-269; F-9 to C-268; F-9 to T-267; F-9 to E-266; F-9 toE-265; F-9 to D-264; F-9 to L-263; F-9 to E-262; F-9 to K-261; F-9 toN-260; F-9 to P-259; F-9 to G-258; F-9 to C-257; F-9 to I-256; F-9 toD-255; F-9 to H-254; F-9 to F-253; F-9 to G-252; F-9 to D-251; F-9 toT-250; F-9 to S-249; F-9 to D-248; F-9 to D-247; F-9 to G-246; F-9 toA-245; F-9 to D-244; F-9 to S-243; F-9 to S-242; F-9 to F-241; F-9 toM-240; F-9 to F-239; F-9 to D-238; F-9 to E-237; F-9 to Q-236; F-9 toA-235; F-9 to L-234; F-9 to C-233; F-9 to R-232; F-9 to C-231; F-9 toI-230; F-9 to H-229; F-9 to N-228; F-9 to N-227; F-9 to W-226; F-9 toM-225; F-9 to Y-224; F-9 to N-223; F-9 to T-222; F-9 to P-221; F-9 toC-220; F-9 to T-219; F-9 to K-218; F-9 to N-217; F-9 to A-216; F-9 toA-215; F-9 to Q-214; F-9 to C-213; F-9 to Q-212; F-9 to P-211; F-9 toL-210; F-9 to T-209; F-9 to A-208; F-9 to P-207; F-9 to L-206; F-9 toS-205; F-9 to R-204; F-9 to R-203; F-9 to I-202; F-9 to I-201; F-9 toS-200; F-9 to H-199; F-9 to V-198; F-9 to Q-197; F-9 to R-196; F-9 toY-195; F-9 to V-194; F-9 to D-193; F-9 to L-192; F-9 to K-191; F-9 toS-190; F-9 to M-189; F-9 to C-188; F-9 to R-187; F-9 to C-186; F-9 toS-185; F-9 to T-184; F-9 to H-183; F-9 to N-182; F-9 to A-181; F-9 toF-180; F-9 to S-179; F-9 to I-178; F-9 to T-177; F-9 to V-176; F-9 toP-175; F-9 to K-174; F-9 to P-173; F-9 to G-172; F-9 to Q-171; F-9 toS-170; F-9 to L-169; F-9 to P-168; F-9 to V-167; F-9 to T-166; F-9 toI-165; F-9 to E-164; F-9 to F-163; F-9 to L-162; F-9 to T-161; F-9 toK-160; F-9 to S-159; F-9 to L-158; F-9 to Y-157; F-9 to S-156; F-9 toT-155; F-9 to S-154; F-9 to T-153; F-9 to N-152; F-9 to M-151; F-9 toC-150; F-9 to Q-149; F-9 to L-148; F-9 to G-147; F-9 to E-146; F-9 toS-145; F-9 to N-144; F-9 to C-143; F-9 to C-142; F-9 to G-141; F-9 toG-140; F-9 to C-139; F-9 to R-138; F-9 to Y-137; F-9 to V-136; F-9 toS-135; F-9 to V-134; F-9 to C-133; F-9 to P-132; F-9 to P-131; F-9 toK-130; F-9 to F-129; F-9 to F-128; F-9 to T-127; F-9 to N-126; F-9 toT-125; F-9 to A-124; F-9 to V-123; F-9 to G-122; F-9 to F-121; F-9 toE-120; F-9 to K-19; F-9 to G-118; F-9 to V-117; F-9 to D-116; F-9 toI-115; F-9 to C-114; F-9 to V-113; F-9 to E-112; F-9 to R-111; F-9 toP-110; F-9 to M-109; F-9 to C-108; F-9 to Q-107; F-9 to T-106; F-9 toK-105; F-9 to R-104; F-9 to W-103; F-9 to E-102; F-9 to N-101; F-9 toD-100; F-9 to I-99; F-9 to S-98; F-9 to K-97; F-9 to L-96; F-9 to I-95;F-9 to E-94; F-9 to T-93; F-9 to N-92; F-9 to Y-91; F-9 to H-90; F-9 toA-89; F-9 to A-88; F-9 to A-87; F-9 to F-86; F-9 to K-85; F-9 to I-84;F-9 to T-83; F-9 to E-82; F-9 to E-81; F-9 to T-80; F-9 to R-79; F-9 toS-78; F-9 to N-77; F-9 to L-76; F-9 to N-75; F-9 to A-74; F-9 to Q-73;F-9 to E-72; F-9 to R-71; F-9 to N-70; F-9 to H-69; F-9 to Q-68; F-9 toW-67; F-9 to G-66; F-9 to G-65; F-9 to K-64; F-9 to R-63; F-9 to L-62;F-9 to Q-61; F-9 to C-60; F-9 to K-59; F-9 to Y-58; F-9 to M-57; F-9 toK-56; F-9 to W-55; F-9 to Y-54; F-9 to E-53; F-9 to P-52; F-9 to Y-51;F-9 to L-50; F-9 to V-49; F-9 to T-48; F-9 to M-47; F-9 to L-46; F-9 toE-45; F-9 to D-44; F-9 to V-43; F-9 to S-42; F-9 to S-41; F-9 to V-40;F-9 to S-39; F-9 to R-38; F-9 to L-37; F-9 to Q-36; F-9 to E-35; F-9 toE-34; F-9 to L-33; F-9 to D-32; F-9 to K-31; F-9 to S-30; F-9 to A-29;F-9 to Y-28; F-9 to A-27; F-9 to T-26; F-9 to A-25; F-9 to E-24; F-9 toG-23; F-9 to A-22; F-9 to D-21; F-9 to P-20; F-9 to E-19; F-9 to A-18;F-9 to D-17; F-9 to S-16; F-9 to L-15; of SEQ ID NO:2. Specificallypreferred is the polypeptide fragment comprising amino acid residues F-9to R-203 of SEQ ID NO:2, as well as polynucleotides encoding thispolypeptide. This F-9 to R-203 of SEQ ID NO:2 polypeptide preferably isassociated with a S-205 to S-396 of SEQ ID NO:2 polypeptide. Associationmay be through disulfide, covalent or noncovalent interactions, bylinkage via a linker (e.g. serine, glycine, proline linkages), or by anantibody.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1660 of SEQID NO: 1, b is an integer of 15 to 1674, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:1, and wherethe b is greater than or equal to a +14.

Thus, in one aspect, N-terminal deletion mutants are provided by thepresent invention. Such mutants include those comprising the amino acidsequence shown in FIG. 1 (SEQ ID NO: 18) except for a deletion of atleast the first 24 N-terminal amino acid residues (i.e., a deletion ofat least Met (1)-Glu (24)) but not more than the first 115 N-terminalamino acid residues of FIG. 1 (SEQ ID NO:18). Alternatively, first 24N-terminal amino acid residues (i.e., a deletion of at least Met (1)-Glu(24)) but not more than the first 103 N-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 18), etc.

In another aspect, C-terminal deletion mutants are provided by thepresent invention. Such mutants include those comprising the amino acidsequence shown in FIG. 1 (SEQ ID NO:18) except for a deletion of atleast the last C-terminal amino acid residue (Ser (419)) but not morethan the last 220 C-terminal amino acid residues (i.e., a deletion ofamino acid residues Val (199)-Ser (419)) of FIG. 1 (SEQ ID NO:18).Alternatively, the deletion will include at least the last C-terminalamino acid residue but not more than the last 216 C-terminal amino acidresidues of FIG. 1 (SEQ ID NO: 18). Alternatively, the deletion willinclude at least the last C-terminal amino acid residue but not morethan the last 204 C-terminal amino acid residues of FIG. 1 (SEQ ID NO:18). Alternatively, the deletion will include at least the lastC-terminal amino acid residues but not more than the last 192 C-terminalamino acid residues of FIG. 1 (SEQ ID NO:18). Alternatively, thedeletion will include at least the last C-terminal amino acid residuesbut not more than the last 156 C-terminal amino acid residues of FIG. 1(SEQ ID NO: 18). Alternatively, the deletion will include at least thelast C-terminal amino acid residues but not more than the last 108C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). Alternatively,the deletion will include at least the last C-terminal amino acidresidues but not more than the last 52 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 18).

In yet another aspect, also included by the present invention aredeletion mutants having amino acids deleted from both the N-terminal andC-terminal residues. Such mutants include all combinations of theN-terminal deletion mutants and C-terminal deletion mutants describedabove.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA(s) or the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3is intended fragments at least about 15 nt, and more preferably at leastabout 20 nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments of 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675,700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025,1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325,1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625,1650 or 1674 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA(s) or as shown in SEQ ID NO:1or SEQ ID NO:3. By a fragment at least 20 nt in length, for example, isintended fragments which include 20 or more contiguous bases from thenucleotide sequence of the deposited cDNA(s) or the nucleotide sequenceas shown in SEQ ID NOS:1 or 3.

Moreover, representative examples of VEGF-2 polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350,351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800,800-850, 851-900, 901-950, or 951 to the end of SEQ ID NO:1 or the cDNAcontained in the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promoter regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

A VEGF-2 “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO:1 or for instance, the cDNA clone(s) contained inATCC Deposit Nos. 97149 or 75698, the complement thereof. “Stringenthybridization conditions” refers to an overnight incubation at 42° C. ina solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

Also contemplated are nucleic acid molecules that hybridize to theVEGF-2 polynucleotides at lower stringency hybridization conditions.Changes in the stringency of hybridization and signal detection areprimarily accomplished through the manipulation of formamideconcentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1). Of course, apolynucleotide which hybridizes only to a poly A sequence (such as the3N terminal poly(A) tract of the VEGF-2 cDNA shown in SEQ ID NOS:1 or3), or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to aportion of a nucleic acid of the invention, since such a polynucleotidewould hybridize to any nucleic acid molecule containing a poly (A)stretch or the complement thereof (e.g., practically any double-strandedcDNA clone).

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin SEQ ID NOS:1 or 3 or to the nucleic acid sequence of the depositedcDNA(s), irrespective of whether they encode a polypeptide having VEGF-2activity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having VEGF-2 activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe or a polymerase chain reaction (PCR)primer. Uses of the nucleic acid molecules of the present invention thatdo not encode a polypeptide having VEGF-2 activity include, inter alia,(1) isolating the VEGF-2 gene or allelic variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theVEGF-2 gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and Northern Blotanalysis for detecting VEGF-2 mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence shown inSEQ ID NOS:1 or 3 or to a nucleic acid sequence of the deposited cDNA(s)which do, in fact, encode a polypeptide having VEGF-2 protein activity.By “a polypeptide having VEGF-2 activity” is intended polypeptidesexhibiting VEGF-2 activity in a particular biological assay. Forexample, VEGF-2 protein activity can be measured using, for example,mitogenic assays and endothelial cell migration assays. See, e.g.,Olofsson et al., Proc. Natl. Acad. Sci. USA 93:2576-2581 (1996) andJoukov et al., EMBO J. 5:290-298 (1996).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence of the depositedcDNA(s) or the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ IDNO:3 will encode a polypeptide “having VEGF-2 protein activity.” Infact, since degenerate variants of these nucleotide sequences all encodethe same polypeptide, this will be clear to the skilled artisan evenwithout performing the above described comparison assay. It will befurther recognized in the art that, for such nucleic acid molecules thatare not degenerate variants, a reasonable number will also encode apolypeptide having VEGF-2 protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95%, 96%, 97%, or 98% identity to a polynucleotide which encodesthe polypeptides of SEQ ID NOS:2 or 4, as well as fragments thereof,which fragments have at least 30 bases and preferably at least 50 basesand to polypeptides encoded by such polynucleotides.

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g.: (COMPUTATIONAL MOLECULARBIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, (1988);BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed.,Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA,PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G.,Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, (1991).) While thereexists a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans. (Carillo, H., and Lipton, D., SIAM J AppliedMath. 48:1073 (1988).) Methods commonly employed to determine identityor similarity between two sequences include, but are not limited to,those disclosed in “Guide to Huge Computers,” Martin J. Bishop, ed.,Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAMJ. Applied Math. 48:1073 (1988). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol.215:403 (1990), Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711 (using the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981)). By a polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theVEGF-2 polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence SEQ ID NO: 1, the ORF (open reading frame), or anyfragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO: 18 or tothe amino acid sequence encoded by deposited DNA clones can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. (1990) 6:237-245). In a sequence alignment the query andsubject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty-0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

VEGF-2 Polypeptides

The present invention further relates to polypeptides which have thededuced amino acid sequence of FIG. 1 or 2, or which has the amino acidsequence encoded by the deposited cDNAs, as well as fragments, analogs,and derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 or 2 or that encoded by the deposited cDNA, meansa polypeptide which retains the conserved motif of VEGF proteins asshown in FIG. 3 and essentially the same biological function oractivity.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be about 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

Preferred polypeptide fragments include the secreted VEGF-2 protein(which is preferably a dimer of amino acids beginning at residue 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114 ofSEQ ID NO:18 and ending at amino acid residue 221, 222, 223, 224, 225,226, 227, 228, or 229 of SEQ ID NO: 18, the pro-protein form (which ispreferably about amino acid 32-419 of SEQ ID NO:18), and mature form.Other preferred polypeptide fragments include the mature form having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of either the secretedVEGF-2 polypeptide or the mature form. Similarly, any number of aminoacids, ranging from I-30, can be deleted from the carboxy terminus ofthe secreted VEGF-2 protein or mature form. Furthermore, any combinationof the above amino and carboxy terminus deletions are preferred.Similarly, polynucleotide fragments encoding these VEGF-2 polypeptidefragments are also preferred.

Also preferred are VEGF-2 polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2 falling withinconserved domains are specifically contemplated by the presentinvention. (See FIG. 2.) Moreover, polynucleotide fragments encodingthese domains are also contemplated.

Other preferred fragments are biologically active VEGF-2 fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the VEGF-2 polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides,preferably recombinant polypeptides.

It will be recognized in the art that some amino acid sequences of theVEGF-2 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

Thus, the invention further includes variations of the VEGF-2polypeptide which show substantial VEGF-2 polypeptide activity or whichinclude regions of VEGF-2 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions. As indicated above, guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin Bowie, J. U., et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

Thus, the fragments, derivatives, or analogs of the polypeptides of FIG.1 or 2, or that encoded by the deposited cDNAs may be: (I) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or (ii) one in which one or more of theamino acid residues includes a substituent group; or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence; or (v) one in which comprisesfewer amino acid residues shown in SEQ ID NOS: 2 or 4, and retains theconserved motif and yet still retains activity characteristics of theVEGF family of polypeptides. Such fragments, derivatives, and analogsare deemed to be within the scope of those skilled in the art from theteachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the VEGF-2 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-a to only one of the two known types of TNF receptors. Thus, theVEGF-2 of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of substitutions for any given VEGF-2polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

Amino acids in the VEGF-2 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

In specific embodiments, the polynucleotides of the invention are lessthan 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.In a further embodiment, polynucleotides of the invention comprise atleast 15 contiguous nucleotides of VEGF-2 coding sequence, but do notcomprise all or a portion of any VEGF-2 intron. In another embodiment,the nucleic acid comprising VEGF-2 coding sequence does not containcoding sequences of a genomic flanking gene (i.e., 5′ or 3′ to theVEGF-2 gene in the genome).

The polypeptides of the present invention include the polypeptides ofSEQ ID NOS:2 and 4 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptides of SEQ ID NOS:2 and 4, and more preferablyat least 90% similarity (more preferably at least 95% identity) to thepolypeptides of SEQ ID NOS:2 and 4, and still more preferably at least95% similarity (still more preferably at least 90% identity) to thepolypeptides of SEQ ID NOS:2 and 4 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The polypeptides of the present invention include the polypeptideencoded by the deposited cDNA including the leader; the maturepolypeptide encoded by the deposited the cDNA minus the leader (i.e.,the mature protein); a polypeptide comprising amino acids about −23 toabout 396 in SEQ ID NO:2; a polypeptide comprising amino acids about −22to about 396 in SEQ ID NO:2; a polypeptide comprising amino acids about1 to about 396 in SEQ ID NO:2; as well as polypeptides which are atleast 95% identical, and more preferably at least 96%, 97%, 98% or 99%identical to the polypeptides described above and also include portionsof such polypeptides with at least 30 amino acids and more preferably atleast 50 amino acids.

Fusion Proteins

Any VEGF-2 polypeptide can be used to generate fusion proteins. Forexample, the VEGF-2 polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the VEGF-2polypeptide can be used to indirectly detect the second protein bybinding to the VEGF-2. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the VEGF-2 polypeptidescan be used as a targeting molecule once fused to other proteins.

Examples of domains that can be fused to VEGF-2 polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the VEGF-2 polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the VEGF-2 polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the VEGF-2polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the VEGF-2 polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acidsI-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-x of human serum albumin, where x is an integerfrom 1 to 585 and the albumin fragment has human serum albumin activity.In another preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric secreted polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above polypeptides canalso be recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto N12+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the VEGF-2 polypeptides can be fused to marker sequences, suchas a peptide which facilitates purification of VEGF-2. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the VEGF-2polynucleotides or the polypeptides.

Biological Activities of VEGF-2

VEGF-2 polynucleotides and polypeptides can be used in assays to testfor one or more biological activities. If VEGF-2 polynucleotides andpolypeptides do exhibit activity in a particular assay, it is likelythat VEGF-2 may be involved in the diseases associated with thebiological activity. Therefore, VEGF-2 or VEGF-2 antibodies could beused to treat the associated disease.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of the antibodiesof the invention. Malignant and metastatic conditions which can betreated with the antibodies of the invention include, but are notlimited to, malignancies, solid tumors, and cancers described herein andotherwise known in the art (for a review of such disorders, see Fishmanet al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount ofan antibody of the invention. For example, antibodies may be utilized ina variety of additional methods in order to therapeutically treat acancer or tumor. Cancers which may be treated with antibodies include,but are not limited to solid tumors, including prostate, lung, breast,brain, ovarian, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, thyroid cancer; primary tumors and metastases;melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-smallcell lung cancer; colorectal cancer; advanced malignancies; and bloodborn tumors such as leukemias. For example, antibodies may be deliveredtopically, in order to treat cancers such as skin cancer, head and necktumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, antibodies may be utilized to treatsuperficial forms of bladder cancer by, for example, intravesicaladministration. Antibodies may be delivered directly into the tumor, ornear the tumor site, via injection or a catheter. Of course, as theartisan of ordinary skill will appreciate, the appropriate mode ofadministration will vary according to the cancer to be treated. Othermodes of delivery are discussed herein.

Antibodies may be useful in treating other disorders, besides cancers,which involve angiogenesis. These disorders include, but are not limitedto: benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; artherosclericplaques; ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) ofthe eye; rheumatoid arthritis; psoriasis; delayed wound healing;endometriosis; vasculogenesis; granulations; hypertrophic scars(keloids); nonunion fractures; scleroderma; trachoma; vascularadhesions; myocardial angiogenesis; coronary collaterals; cerebralcollaterals; arteriovenous malformations; ischemic limb angiogenesis;Osler-Webber Syndrome; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; fibromuscular dysplasia; woundgranulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering a antibodies of the invention to a hypertrophicscar or keloid.

Within one embodiment of the present invention antibodies of theinvention are directly injected into a hypertrophic scar or keloid, inorder to prevent the progression of these lesions. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development. As notedabove, the present invention also provides methods for treatingneovascular diseases of the eye, including for example, cornealneovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

Moreover, ocular disorders associated with neovascularization which canbe treated with the antibodies of the present invention include, but arenot limited to: neovascular glaucoma, diabetic retinopathy,retinoblastoma, retrolental fibroplasia, uveitis, retinopathy ofprematurity macular degeneration, corneal graft neovascularization, aswell as other eye inflammatory diseases, ocular tumors and diseasesassociated with choroidal or iris neovascularization. See, e.g., reviewsby Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al.,Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above, includingantibodies) to the cornea, such that the formation of blood vessels isinhibited. Briefly, the cornea is a tissue which normally lacks bloodvessels. In certain pathological conditions however, capillaries mayextend into the cornea from the pericorneal vascular plexus of thelimbus. When the cornea becomes vascularized, it also becomes clouded,resulting in a decline in the patient's visual acuity. Visual loss maybecome complete if the cornea completely opacitates. A wide variety ofdisorders can result in corneal neovascularization, including forexample, corneal infections (e.g., trachoma, herpes simplex keratitis,leislmaniasis and onchocerciasis), immunological processes (e.g., graftrejection and Stevens-Johnson's syndrome), alkali burns, trauma,inflammation (of any cause), toxic and nutritional deficiency states,and as a complication of wearing contact lenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical burns). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the antibodies described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of an antibody to the eye,such that the formation of blood vessels is inhibited. In oneembodiment, the compound may be administered topically to the eye inorder to treat early forms of neovascular glaucoma. Within otherembodiments, the compound may be implanted by injection into the regionof the anterior chamber angle. Within other embodiments, the compoundmay also be placed in any location such that the compound iscontinuously released into the aqueous humor. Within another aspect ofthe present invention, methods are provided for treating proliferativediabetic retinopathy, comprising the step of administering to a patienta therapeutically effective amount of a an antibody to the eyes, suchthat the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the antibodies in the retina. Preferably, thistreatment should be initiated prior to the acquisition of severe diseaserequiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of an antibody to theeye, such that the formation of blood vessels is inhibited. The compoundmay be administered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, disorders which can be treated with antibodies include,but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated, prevented,diagnosed, and/or prognosed with the antibodies of the inventioninclude, but are not limited to, solid tumors, blood born tumors such asleukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayedwound healing, endometriosis, vascluogenesis, granulations, hypertrophicscars (keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method. Antibodiesmay also be used in controlling menstruation or administered as either aperitoneal lavage fluid or for peritoneal implantation in the treatmentof endometriosis.

Antibodies of the present invention may be incorporated into surgicalsutures in order to prevent stitch granulomas.

Antibodies may be utilized in a wide variety of surgical procedures. Forexample, within one aspect of the present invention a compositions (inthe form of, for example, a spray or film) may be utilized to coat orspray an area prior to removal of a tumor, in order to isolate normalsurrounding tissues from malignant tissue, and/or to prevent the spreadof disease to surrounding tissues. Within other aspects of the presentinvention, compositions (e.g., in the form of a spray) may be deliveredvia endoscopic procedures in order to coat tumors, or inhibitangiogenesis in a desired locale. Within yet other aspects of thepresent invention, surgical meshes which have been coated withanti-angiogenic compositions of the present invention may be utilized inany procedure wherein a surgical mesh might be utilized. For example,within one embodiment of the invention a surgical mesh laden with ananti-angiogenic composition may be utilized during abdominal cancerresection surgery (e.g., subsequent to colon resection) in order toprovide support to the structure, and to release an amount of theanti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering an antibodyto the resection margins of a tumor subsequent to excision, such thatthe local recurrence of cancer and the formation of new blood vessels atthe site is inhibited. Within one embodiment of the invention, theanti-angiogenic compound, for example VEGF-2 antibody, is administereddirectly to the tumor excision site (e.g., applied by swabbing, brushingor otherwise coating the resection margins of the tumor with the VEGF-2antibody). Alternatively, the VEGF-2 antibodies may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, VEGF-2 antibodies are appliedafter hepatic resections for malignancy, and after neurosurgicaloperations.

Within one aspect of the present invention, VEGF-2 antibodies may beadministered to the resection margin of a wide variety of tumors,including for example, breast, colon, brain and hepatic tumors. Forexample, within one embodiment of the invention, VEGF-2 antibodies maybe administered to the site of a neurological tumor subsequent toexcision, such that the formation of new blood vessels at the site areinhibited.

Antibodies of the present invention may also be administered along withother anti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; andmetalloproteinase inhibitors such as BB94.

Immune Activity

VEGF-2 antibodies may be useful in treating deficiencies or disorders ofthe immune system, by activating or inhibiting the proliferation,differentiation, or mobilization (chemotaxis) of immune cells. Immunecells develop through a process called hematopoiesis, producing myeloid(platelets, red blood cells, neutrophils, and macrophages) and lymphoid(B and T lymphocytes) cells from pluripotent stem cells. The etiology ofthese immune deficiencies or disorders may be genetic, somatic, such ascancer or some autoimmune disorders, acquired (e.g., by chemotherapy ortoxins), or infectious. Moreover, VEGF-2 antibodies can be used as amarker or detector of a particular immune system disease or disorder.

VEGF-2 antibodies may be useful in treating or detecting deficiencies ordisorders of hematopoietic cells. VEGF-2 antibodies could be used toincrease differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat thosedisorders associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein disorders (e.g.agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, commonvariable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLVinfection, leukocyte adhesion deficiency syndrome, lymphopenia,phagocyte bactericidal dysfunction, severe combined immunodeficiency(SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, orhemoglobinuria.

Moreover, VEGF-2 antibodies can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, VEGF-2antibodies could be used to treat blood coagulation disorders (e.g.,afibrinogenemia, factor deficiencies), blood platelet disorders (e.g.thrombocytopenia), or wounds resulting from trauma, surgery, or othercauses. Alternatively, VEGF-2 antibodies that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting,important in the treatment of heart attacks (infarction), strokes, orscarring.

VEGF-2 antibodies may also be useful in treating or detecting autoimmunedisorders. Many autoimmune disorders result from inappropriaterecognition of self as foreign material by immune cells. Thisinappropriate recognition results in an immune response leading to thedestruction of the host tissue. Therefore, the administration of VEGF-2antibodies that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected byVEGF-2 antibodies include, but are not limited to: Addison's Disease,hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by VEGF-2 antibodies. Moreover, VEGF-2 antibodies can be usedto treat anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

VEGF-2 antibodies may also be used to treat and/or prevent organrejection or graft-versus-host disease (GVHD). Organ rejection occurs byhost immune cell destruction of the transplanted tissue through animmune response. Similarly, an immune response is also involved in GVHD,but, in this case, the foreign transplanted immune cells destroy thehost tissues. The administration of VEGF-2 antibodies that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, VEGF-2 antibodies may also be used to modulate inflammation.For example, VEGF-2 antibodies may inhibit the proliferation anddifferentiation of cells involved in an inflammatory response. Thesemolecules can be used to treat inflammatory conditions, both chronic andacute conditions, including inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory response syndrome(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

VEGF-2 antibodies of the invention, can be used to treat or detecthyperproliferative disorders, including neoplasms. VEGF-2 antibodies mayinhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, VEGF-2 antibodies may proliferate othercells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby VEGF-2 antibodies include, but are not limited to neoplasms locatedin the: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, brain, nervous(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, prostate, thoracic, and urogenital. In preferred embodiments,VEGF-2 antibodies may be used to treat, prevent or ameliorate breastcancer. In other preferred embodiments, VEGF-2 antibodies may be used totreat, prevent or ameliorate brain cancer. In other preferredembodiments, VEGF-2 antibodies may be used to treat, prevent orameliorate prostate cancer. In other preferred embodiments, VEGF-2antibodies may be used to treat, prevent or ameliorate colon cancer. Inother preferred embodiments, VEGF-2 antibodies may be used to treat,prevent or ameliorate Kaposi's sarcoma.

Similarly, other hyperproliferative disorders can also be treated ordetected by VEGF-2 antibodies. Examples of such hyperproliferativedisorders include, but are not limited to: hypergammaglobulinemia,lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis,Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease,histiocytosis, and any other hyperproliferative disease, besidesneoplasia, located in an organ system listed above.

Binding Activity

VEGF-2 polypeptides may be used to screen for molecules that bind toVEGF-2 or for molecules to which VEGF-2 binds. The binding of VEGF-2 andthe molecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the VEGF-2 or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand ofVEGF-2, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which VEGF-2binds (i.e., flk-1 or flt-4), or at least, a fragment of the receptorcapable of being bound by VEGF-2 (e.g., active site). In either case,the molecule can be rationally designed using known techniques.Preferably, the screening for these molecules involves producingappropriate cells which express VEGF-2, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing VEGF-2 (or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either VEGF-2 or the molecule.

The assay may simply test binding of a candidate compound to VEGF-2,wherein binding is detected by a label, or in an assay involvingcompetition with a labeled competitor. Further, the assay may testwhether the candidate compound results in a signal generated by bindingto VEGF-2.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining VEGF-2, measuring VEGF-2/molecule activity or binding, andcomparing the VEGF-2/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure VEGF-2 level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure VEGF-2 level or activity by eitherbinding, directly or indirectly, to VEGF-2 or by competing with VEGF-2for a substrate.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting the VEGF-2/molecule.Moreover, the assays can discover agents which may inhibit or enhancethe production of VEGF-2 from suitably manipulated cells or tissues.

Therefore, the invention includes a method of identifying compoundswhich bind to VEGF-2 comprising the steps of: (a) incubating a candidatebinding compound with VEGF-2; and (b) determining if binding hasoccurred. Moreover, the invention includes a method of identifyingagonists/antagonists comprising the steps of: (a) incubating a candidatecompound with VEGF-2, (b) assaying a biological activity, and (b)determining if a biological activity of VEGF-2 has been altered.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for VEGF-2polypeptides, or cells expressing a cell bound form of VEGF-2polypeptide.

As discussed herein, antibodies of the invention may be associated withheterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering antibodies of the invention that are associated withheterologous polypeptides or nucleic acids. In one example, theinvention provides a method for delivering a therapeutic protein intothe targeted cell. In another example, the invention provides a methodfor delivering a single stranded nucleic acid (e.g., antisense orribozymes) or double stranded nucleic acid (e.g., DNA that can integrateinto the cell's genome or replicate episomally and that can betranscribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering VEGF-2 antibodies of the invention in association withtoxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of VEGF-2 polypeptides. Such amethod would include contacting the polypeptide of the present inventionwith a selected compound(s) suspected of having antagonist or agonistactivity, and assaying the activity of these polypeptides followingbinding.

This invention is particularly useful for screening therapeuticcompounds by using the VEGF-2 polypeptides, or binding fragmentsthereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by VEGF-2polypeptides. These methods comprise contacting such an agent with aVEGF-2 polypeptide or a fragment thereof and assaying for the presenceof a complex between the agent and the polypeptide or a fragmentthereof, by methods well known in the art. In such a competitive bindingassay, the agents to screen are typically labeled. Following incubation,free agent is separated from that present in bound form, and the amountof free or uncomplexed label is a measure of the ability of a particularagent to bind to VEGF-2 polypeptides.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to VEGF-2 polypeptides,and is described in great detail in European Patent Application84/03564, published on Sep. 13, 1984, which is incorporated herein byreference herein. Briefly stated, large numbers of different smallpeptide test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The peptide test compounds arereacted with polypeptides of the present invention and washed. Boundpolypeptides are then detected by methods well known in the art.Purified polypeptides are coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies may be used to capture the peptide and immobilize it on thesolid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding VEGF-2polypeptides specifically compete with a test compound for binding tothe VEGF-2 polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind VEGF-2 polypeptides, and thebinding molecules identified thereby. These binding molecules areuseful, for example, as agonists and antagonists, such as VEGF-2antibodies, of the VEGF-2 polypeptides. Such antibodies can be used, inaccordance with the invention, in the therapeutic embodiments describedin detail, below.

This method comprises the steps of: contacting VEGF-2 polypeptides witha plurality of molecules; and identifying a molecule that binds VEGF-2polypeptides.

The step of contacting VEGF-2 polypeptides with the plurality ofmolecules may be effected in a number of ways. For example, one maycontemplate immobilizing the VEGF-2 polypeptides on a solid support andbringing a solution of the plurality of molecules in contact with theimmobilized VEGF-2 polypeptides. Such a procedure would be akin to anaffinity chromatographic process, with the affinity matrix beingcomprised of the immobilized polypeptides of the invention. Themolecules having a selective affinity for the VEGF-2 polypeptides canthen be purified by affinity selection. The nature of the solid support,process for attachment of the polypeptides to the solid support,solvent, and conditions of the affinity isolation or selection arelargely conventional and well known to those of ordinary skill in theart.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the polypeptides of the invention, optionally in thepresence of an inducer should one be required for expression, todetermine if any selective affinity interaction takes place between thepolypeptides and the individual clone. Prior to contacting the VEGF-2polypeptides with each fraction comprising individual polypeptides, thepolypeptides could first be transferred to a solid support foradditional convenience. Such a solid support may simply be a piece offilter membrane, such as one made of nitrocellulose or nylon. In thismanner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity forpolypeptides of the invention. Furthermore, the amino acid sequence ofthe polypeptide having a selective affinity for the polypeptides of theinvention can be determined directly by conventional means or the codingsequence of the DNA encoding the polypeptide can frequently bedetermined more conveniently. The primary sequence can then be deducedfrom the corresponding DNA sequence. If the amino acid sequence is to bedetermined from the polypeptide itself, one may use microsequencingtechniques. The sequencing technique may include mass spectroscopy.

In certain situations, it may be desirable to wash away any unboundpolypeptides from a mixture of the VEGF-2 polypeptides and the pluralityof polypeptides prior to attempting to determine or to detect thepresence of a selective affinity interaction. Such a wash step may beparticularly desirable when the VEGF-2 polypeptides or the plurality ofpolypeptides are bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically VEGF-2 polypeptides. Many libraries are known in the artthat can be used, e.g., chemically synthesized libraries, recombinant(e.g., phage display libraries), and in vitro translation-basedlibraries. Examples of chemically synthesized libraries are described inFodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994,Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618;Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCTPublication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl.Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CTPublication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindsVEGF-2 polypeptides can be carried out by contacting the library memberswith polypeptides of the invention immobilized on a solid phase andharvesting those library members that bind to VEGF-2 polypeptides.Examples of such screening methods, termed “panning” techniques aredescribed by way of example in Parmley and Smith, 1988, Gene 73:305-318;Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO94/18318; and in references cited herein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to VEGF-2 polypeptides.

Where the binding molecule is a polypeptide, the polypeptide can beconveniently selected from any peptide library, including random peptidelibraries, combinatorial peptide libraries, or biased peptide libraries.The term “biased” is used herein to mean that the method of generatingthe library is manipulated so as to restrict one or more parameters thatgovern the diversity of the resulting collection of molecules, in thiscase peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a binding polypeptide has in the range of 15-100 aminoacids, or 20-50 amino acids.

The selected binding polypeptide can be obtained by chemical synthesisor recombinant expression.

Vectors, Host Cells, and Protein Production

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofVEGF-2 polypeptides or peptides by recombinant techniques.

Host cells are genetically engineered (transduced, transformed, ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the VEGF-2 genes of the invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide sequence may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids;vectors derived from combinations of plasmids and phage DNA, viral DNAsuch as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However,any other plasmid or vector may be used so long as it is replicable andviable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain at least oneselectable marker gene to provide a phenotypic trait for selection oftransformed host cells. Such markers include dihydrofolate reductase(DHFR) or neomycin resistance for eukaryotic cell culture, andtetracycline or ampicillin resistance for culturing in E. coli and otherbacteria.

The vector containing the appropriate. DNA sequence as herein abovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. Representative examples of appropriate hosts,include but are not limited to: bacterial cells, such as E. coli,Salmonella typhimurium, and Streptomyces; fungal cells, such as yeast;insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, and Bowes melanoma; and plant cells. The selection ofan appropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example—bacterial: pQE70, pQE60 and pQE-9,available from Qiagen; pBS vectors, Phagescript vectors, Bluescriptvectors, pNH8A, pNH16a, pNH118A, pNH46A, available from Stratagene; andptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 andpSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

In addition to the use of expression vectors in the practice of thepresent invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4a which is described in detail below.

As summarized in FIGS. 28 and 29, components of the pHE4a vector (SEQ IDNO: 16) include: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,6) the lactose operon repressor gene (lacIq) and 7) a multiple cloningsite linker region. The origin of replication (oriC) is derived frompUC19 (LTI, Gaithersburg, Md.). The promoter sequence and operatorsequences were made synthetically. Synthetic production of nucleic acidsequences is well known in the art. CLONTECH 95/96 Catalog, pages215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303. ThepHE4a vector was deposited with the ATCC on Feb. 25, 1998, and givenaccession number 209645.

A nucleotide sequence encoding VEGF-2 (SEQ ID NO: 1), is operativelylinked to the promoter and operator of pHE4a by restricting the vectorwith NdeI and either XbaI, BamHI, XhoI, or Asp718, and isolating thelarger fragment (the multiple cloning site region is about 310nucleotides) on a gel. The nucleotide sequence encoding VEGF-2 (SEQ IDNO:1) having the appropriate restriction sites is generated, forexample, according to the PCR protocol described in Example 1, using PCRprimers having restriction sites for NdeI (as the 5′ primer) and eitherXbaI, BamHI, XhoI, or Asp718 (as the 3′ primer). The PCR insert is gelpurified and restricted with compatible enzymes. The insert and vectorare ligated according to standard protocols.

As noted above, the pHE4a vector contains a lacIq gene. LacIq is anallele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene51:255-267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). VEGF-2 thusis not produced in appreciable quantities in uninduced host cellscontaining the pHE4a vector. Induction of these host cells by theaddition of an agent such as IPTG, however, results in the expression ofthe VEGF-2 coding sequence.

The promoter/operator sequences of the pHE4a vector (SEQ ID NO: 17)comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the lacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages802-807.

The pHE4 series of vectors contain all of the components of the pHE4avector except for the VEGF-2 coding sequence. Features of the pHE4avectors include optimized synthetic T5 phage promoter, lac operator, andShine-Delagarno sequences. Further, these sequences are also optimallyspaced so that expression of an inserted gene may be tightly regulatedand high level of expression occurs upon induction.

Among known bacterial promoters suitable for use in the production ofproteins of the present invention include the E. coli lacI and lacZpromoters, the T3 and T7 promoters, the gpt promoter, the lambda PR andPL promoters and the trp promoter. Suitable eukaryotic promoters includethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus (RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter.

The pHE4a vector also contains a Shine-Delgarno sequence 5′ to the AUGinitiation codon. Shine-Delgarno sequences are short sequences generallylocated about 10 nucleotides up-stream (i.e., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon.

Thus, the present invention is also directed to expression vector usefulfor the production of the proteins of the present invention. This aspectof the invention is exemplified by the pHE4a vector (SEQ ID NO: 16).

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, electroporation, transduction, infection, or other methods(Davis, L., et al, Basic Methods in Molecular Biology (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook. et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989), the disclosure of which is herebyincorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, well known to those skilled in the art, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., VEGF-2 sequence), and/or to include geneticmaterial (e.g., heterologous promoters) that is operably associated withVEGF-2 sequence of the invention, and which activates, alters, and/oramplifies endogenous VEGF-2 polynucleotides. For example, techniquesknown in the art may be used to operably associate heterologous controlregions and endogenous polynucleotide sequences (e.g. encoding VEGF-2)via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication No. WO 96/29411, published Sep.26, 1996; International Publication No. WO 94/12650, published Aug. 4,1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

The host cell can be a higher eukaryotic cell, such as a mammalian cell(e.g., a human derived cell), or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. The host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., glycosylation, phosphorylation, cleavage) ofproteins. Appropriate cell lines can be chosen to ensure the desiredmodifications and processing of the protein expressed.

The polypeptides can be recovered and purified from recombinant cellcultures by methods used heretofore, including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of the matureprotein. Finally, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). For example, apeptide corresponding to a fragment of the VEGF-2 polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the VEGF-2polynucleotide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses VEGF-2 polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofVEGF-2 which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The VEGF-2 polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of the VEGF-2polypeptides of the invention, their preparation, and compositions(preferably, pharmaceutical compositions) containing them. In specificembodiments, the polypeptides of the invention are monomers, dimers,trimers or tetramers. In additional embodiments, the multimers of theinvention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyVEGF-2 polypeptides of the invention (including VEGF-2 fragments,variants, splice variants, and fusion proteins, as described herein).These homomers may contain VEGF-2 polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only VEGF-2 polypeptides havingan identical amino acid sequence. In another specific embodiment, ahomomer of the invention is a multimer containing VEGF-2 polypeptideshaving different amino acid sequences. In specific embodiments, themultimer of the invention is a homodimer (e.g., containing VEGF-2polypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing VEGF-2 polypeptides having identical and/ordifferent amino acid sequences). In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the VEGF-2 polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the VEGF-2 polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptide encoded by the depositedclone.) In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a VEGF-2 fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a VEGF-2-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another TNF familyligand/receptor member that is capable of forming covalently associatedmultimers, such as for example, oseteoprotegerin (see, e.g.,International Publication No. WO 98/49305, the contents of which areherein incorporated by reference in its entirety).

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Therapeutic Uses

The VEGF-2 polypeptide of the present invention is a potent mitogen forvascular and lymphatic endothelial cells. As shown in FIGS. 12 and 13,the VEGF-2 polypeptide of SEQ ID NO:2, minus the initial 46 amino acids,is a potent mitogen for vascular endothelial cells and stimulates theirgrowth and proliferation. The results of a Northern blot analysisperformed for the VEGF-2 nucleic acid sequence encoding this polypeptidewherein 20 mg of RNA from several human tissues were probed with³²P-VEGF-2, illustrates that this protein is actively expressed in theheart and lung which is further evidence of mitogenic activity.

VEGF-2 agonistic antibodies, which enhance the activity of VEGF-2, maybe employed to treat vascular trauma by promoting angiogenesis. Forexample, to stimulate the growth of transplanted tissue where coronarybypass surgery is performed. The VEGF-2 agonistic antibodies may also beemployed to promote wound healing, particularly to re-vascularizedamaged tissues or stimulate collateral blood flow during ischemia andwhere new capillary angiogenesis is desired. Such antibodies can beemployed to treat full-thickness wounds such as dermal ulcers, includingpressure sores, venous ulcers, and diabetic ulcers. In addition, VEGF-2agonistic antibodies, may be employed to treat full-thickness burns andinjuries where a skin graft or flap is used to repair such burns andinjuries. Furthermore, the VEGF-2 agonistic antibodies of the inventionmay be employed for use in plastic surgery, for example, for the repairof lacerations, burns, or other trauma. The VEGF-2 agonistic antibodiesof the invention can also be used to promote healing of wounds andinjuries to the eye as well as to treat eye diseases.

Along these same lines, agonistic antibodies can also be used to inducethe growth of damaged bone, periodontium or ligament tissue. Such VEGF-2agonistic antibodies may also be employed for regenerating supportingtissues of the teeth, including cementum and periodontal ligament, thathave been damaged by, e.g., periodontal disease or trauma.

Since angiogenesis is important in keeping wounds clean andnon-infected, agonistic antibodies that enhance the activity of VEGF-2,may be employed in association with surgery and following the repair ofincisions and cuts. These VEGF-2 antibodies may also be employed for thetreatment of abdominal wounds where there is a high risk of infection.

VEGF-2 agonistic antibodies may be employed for the promotion ofendothelialization in vascular graft surgery. In the case of vasculargrafts using either transplanted or synthetic material, these antibodiescan be applied to the surface of the graft or at the junction to promotethe growth of vascular endothelial cells. Antibodies acting as agonistsof VEGF-2 can be employed to repair damage of myocardial tissue as aresult of myocardial infarction and may also be employed to repair thecardiac vascular system after ischemia. VEGF-2 agonistic antibodies mayalso be employed to treat damaged vascular tissue as a result ofcoronary artery disease and peripheral and CNS vascular disease.

VEGF-2 agonistic antibodies may also be employed to coat artificialprostheses or natural organs which are to be transplanted in the body tominimize rejection of the transplanted material and to stimulatevascularization of the transplanted materials.

Agonistic antibodies of VEGF-2 may also be employed for vascular tissuerepair of injuries resulting from trauma, for example, that occurringduring arteriosclerosis and required following balloon angioplasty wherevascular tissues are damaged.

VEGF-2 agonistic antibodies may also be used to treat peripheralarterial disease. Accordingly, in a further aspect, there is provided aprocess for utilizing VEGF-2 agonistic antibodies to treat peripheralarterial disease. Preferably, a VEGF-2 agonistic antibody isadministered to an individual for the purpose of alleviating or treatingperipheral arterial disease. Suitable doses, formulations, andadministration routes are described below.

Agonistic antibodies of VEGF-2 can promote the endothelial function oflymphatic tissues and vessels, such as to treat the loss of lymphaticvessels, and occlusions of lymphatic vessels. Agonistic antibodies ofVEGF-2 may also be used to stimulate lymphocyte production. AntagonisticVEGF-2 antibodies may be used to treat lymphangiomas.

Agonistic antibodies may also be used to treat hemangioma in newborns.Accordingly, in a further aspect, there is provided a process forutilizing VEGF-2 antibodies to treat hemangioma in newborns. Preferably,an antibody is administered to an individual for the purpose ofalleviating or treating hemangioma in newborns. Suitable doses,formulations, and administration routes are described below.

VEGF-2 agonistic antibodies may also be used to prevent or treatabnormal retinal development in premature newborns. Accordingly, in afurther aspect, there is provided a process for utilizing VEGF-2antibodies to treat abnormal retinal development in premature newborns.Preferably, a VEGF-2 antibody is administered to an individual for thepurpose of alleviating or treating abnormal retinal development inpremature newborns. Suitable doses, formulations, and administrationroutes are described below.

VEGF-2 agonistic antibodies may be used to treat primary (idiopathic)lymphedemas, including Milroy's disease and Lymphedema praecox.Preferably, a VEGF-2 antibody is administered to an individual for thepurpose of alleviating or treating primary (idiopathic) lymphedemas,including Milroy's disease and Lymphedema praecox. VEGF-2 antibodies mayalso be used to treat edema as well as to effect blood pressure in ananimal. Suitable doses, formulations, and administration routes aredescribed below.

VEGF-2 agonistic antibodies may also be used to treat secondary(obstructive) lymphedema including those that result from (I) theremoval of lymph nodes and vessels, (ii) radiotherapy and surgery in thetreatment of cancer, and (iii) trauma and infection. Preferably, aVEGF-2 antibody is administered to an individual for the purpose ofsecondary (obstructive) lymphedema including those that result from (I)the removal of lymph nodes and vessels, (ii) radiotherapy and surgery inthe treatment of cancer, and (iii) trauma and infection. Suitable doses,formulations, and administration routes are described below.

VEGF-2 agonistic antibodies may also be used to treat Kaposi's Sarcoma.Preferably, a VEGF-2 antibody is administered to an individual for thepurpose of alleviating or treating Kaposi's Sarcoma. Suitable doses,formulations, and administration routes are described below.

VEGF-2 antibodies can be used to treat cancer by inhibiting theangiogenesis necessary to support cancer and tumor growth.

Cardiovascular Disorders

The present inventors have shown that VEGF-2 stimulates the growth ofvascular endothelial cells, stimulates endothelial cell migration,stimulates angiogenesis in the CAM assay, decreases blood pressure inspontaneously hypertensive rats, and increases blood flow to ischemiclimbs in rabbits. Accordingly, agonistic antibodies that enhance theactivity of VEGF-2 may be used to treat cardiovascular disorders,including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stepalsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

VEGF-2 antibodies, which enhance the activity of VEGF-2, are especiallyeffective for the treatment of critical limb ischemia and coronarydisease. As shown in Example 18, administration of VEGF-2polynucleotides and polypeptides to an experimentally induced ischemiarabbit hind limb restored blood pressure ratio, blood flow, angiographicscore, and capillary density.

Antibodies may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. VEGF-2 antibodies may be administered as part of apharmaceutical composition, described in more detail below. Methods ofdelivering VEGF-2 antibodies are described in more detail below.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of the VEGF-2polypeptide of the present invention. This method requires apolynucleotide which codes for a VEGF-2 polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aVEGF-2 polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the VEGF-2 polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The VEGF-2polynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the VEGF-2 polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the VEGF-2 polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The VEGF-2 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of VEGF-2 DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forVEGF-2.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The VEGF-2 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked acid sequence injection, an effective dosage amount of DNAor RNA will be in the range of from about 0.05 mg/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked VEGF-2 DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

As is evidenced by Example 18, naked VEGF-2 nucleic acid sequences canbe administered in vivo results in the successful expression of VEGF-2polypeptide in the femoral arteries of rabbits.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the VEGF-2 polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15 EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication No. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication No. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are be engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding VEGF-2. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14×, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding VEGF-2. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express VEGF-2.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with VEGF-2 polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses VEGF-2, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis. 109:233-238). Finally, adenovirus mediated gene transferhas been demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The VEGF-2 polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe VEGF-2 polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the VEGF-2 polynucleotide construct integrated intoits genome, and will express VEGF-2.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding VEGF-2) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the VEGF-2 desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous VEGF-2 sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous VEGF-2 sequence.

The polynucleotides encoding VEGF-2 may be administered along with otherpolynucleotides encoding other angiongenic proteins. Angiogenic proteinsinclude, but are not limited to, acidic and basic fibroblast growthfactors, VEGF-1, epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding VEGF-2 contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Nucleic Acid Utilities

VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may also beemployed for in vitro purposes related to scientific research, synthesisof DNA and manufacture of DNA vectors, and for the production ofdiagnostics and therapeutics to treat human disease. For example, VEGF-2may be employed for in vitro culturing of vascular endothelial cells,where it is added to the conditional medium in a concentration from 10pg/ml to 10 ng/ml.

Fragments of the full length VEGF-2 gene may be used as a hybridizationprobe for a cDNA library to isolate other genes which have a highsequence similarity to the gene or similar biological activity. Probesof this type generally have at least 50 base pairs, although they mayhave a greater number of bases. The probe may also be used to identify acDNA clone corresponding to a full length transcript and a genomic cloneor clones that contain the complete VEGF-2 gene including regulatory andpromoter regions, exons, and introns. An example of a screen comprisesisolating the coding region of the VEGF-2 gene by using the known DNAsequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

This invention provides methods for identification of VEGF-2 receptors.The gene encoding the receptor can be identified by numerous methodsknown to those of skill in the art, for example, ligand panning and FACSsorting (Coligan et al., Current Protocols in Immun., 1(2), Chapter 5,(1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to VEGF-2, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to VEGF-2.Transfected cells which are grown on glass slides are exposed to labeledVEGF-2. VEGF-2 can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clone that encodes theputative receptor.

As an alternative approach for receptor identification, labeled VEGF-2can be photoaffinity linked with cell membrane or extract preparationsthat express the receptor molecule. Cross-linked material is resolved byPAGE and exposed to X-ray film. The labeled complex containing VEGF-2 isthen excised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the gene encoding the putativereceptor.

VEGF-2 Agonist and Antagonists

This invention is also related to a method of screening compounds toidentify those which are VEGF-2 agonists or antagonists. An example ofsuch a method takes advantage of the ability of VEGF-2 to significantlystimulate the proliferation of human endothelial cells in the presenceof the comitogen Con A. Endothelial cells are obtained and cultured in96-well flat-bottomed culture plates (Costar, Cambridge, Mass.) in areaction mixture supplemented with Con-A (Calbiochem, La Jolla, Calif.).Con-A, polypeptides of the present invention and the compound to bescreened are added. After incubation at 37 degrees C., cultures arepulsed with 1 FCi of ³-[H]thymidine (5 Ci/mmol; 1 Ci=37 BGq; NEN) for asufficient time to incorporate the ³[H] and harvested onto glass fiberfilters (Cambridge Technology, Watertown, Mass.). Mean ³[H]-thymidineincorporation (cpm) of triplicate cultures is determined using a liquidscintillation counter (Beckman Instruments, Irvine, Calif.). Significant³[H]thymidine incorporation, as compared to a control assay where thecompound is excluded, indicates stimulation of endothelial cellproliferation.

To assay for antagonists, the assay described above is performed and theability of the compound to inhibit ³[H]thymidine incorporation in thepresence of VEGF-2 indicates that the compound is an antagonist toVEGF-2. Alternatively, VEGF-2 antagonists may be detected by combiningVEGF-2 and a potential antagonist with membrane-bound VEGF-2 receptorsor recombinant receptors under appropriate conditions for a competitiveinhibition assay. VEGF-2 can be labeled, such as by radioactivity, suchthat the number of VEGF-2 molecules bound to the receptor can determinethe effectiveness of the potential antagonist.

Alternatively, the response of a known second messenger system followinginteraction of VEGF-2 and receptor would be measured and compared in thepresence or absence of the compound. Such second messenger systemsinclude but are not limited to, cAMP guanylate cyclase, ion channels orphosphoinositide hydrolysis. In another method, a mammalian cell ormembrane preparation expressing the VEGF-2 receptor is incubated withlabeled VEGF-2 in the presence of the compound. The ability of thecompound to enhance or block this interaction could then be measured.

Potential VEGF-2 antagonists include an antibody, or in some cases, anoligonucleotide, which binds to the polypeptide and effectivelyeliminates VEGF-2 function. Alternatively, a potential antagonist may bea closely related protein which binds to VEGF-2 receptors, however, theyare inactive forms of the polypeptide and thereby prevent the action ofVEGF-2. Examples of these antagonists include a dominant negative mutantof the VEGF-2 polypeptide, for example, one chain of the hetero-dimericform of VEGF-2 may be dominant and may be mutated such that biologicalactivity is not retained. An example of a dominant negative mutantincludes truncated versions of a dimeric VEGF-2 which is capable ofinteracting with another dimer to form wild type VEGF-2, however, theresulting homo-dimer is inactive and fails to exhibit characteristicVEGF activity.

Another potential VEGF-2 antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al.,Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)),thereby preventing transcription and the production of VEGF-2. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the VEGF-2 polypeptide(Antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of VEGF-2.

Potential VEGF-2 antagonists also include small molecules which bind toand occupy the active site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

The antagonists, such as VEGF-2 antibodies, may be employed to limitangiogenesis necessary for solid tumor metastasis. The identification ofVEGF-2 can be used for the generation of certain inhibitors of vascularendothelial growth factor. Since angiogenesis and neovascularization areessential steps in solid tumor growth, inhibition of angiogenic activityof the vascular endothelial growth factor is very useful to prevent thefurther growth, retard, or even regress solid tumors. Although the levelof expression of VEGF-2 is extremely low in normal tissues includingbreast, it can be found expressed at moderate levels in at least twobreast tumor cell lines that are derived from malignant tumors. It is,therefore, possible that VEGF-2 is involved in tumor angiogenesis andgrowth.

Gliomas are also a type of neoplasia which may be treated with theantagonists of the present invention.

The antagonists, such as VEGF-2 antibodies, may also be used to treatchronic inflammation caused by increased vascular permeability. Inaddition to these disorders, the antagonists may also be employed totreat retinopathy associated with diabetes, rheumatoid arthritis andpsoriasis.

The antagonists, such as VEGF-2 antibodies, may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

Pharmaceutical Compositions

The VEGF-2 antibodies may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the polypeptide or agonist or antagonist, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intraperitoneal,intramuscular, intratumor, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, the pharmaceutical compositions are administeredin an amount of at least about 10 mg/kg body weight and in most casesthey will be administered in an amount not in excess of about 8 mg/Kgbody weight per day. In most cases, the dosage is from about 10 mg/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

The VEGF-2 antibodies which are polypeptides may also be employed inaccordance with the present invention by expression of such polypeptidein vivo, which is often referred to as “gene therapy,” described above.

Thus, for example, cells such as bone marrow cells may be engineeredwith a polynucleotide (DNA or RNA) encoding for the polypeptide, or anVEGF-2 antibody, ex vivo, the engineered cells are then provided to apatient to be treated with the polypeptide. Such methods are well-knownin the art. For example, cells may be engineered by procedures known inthe art by use of a retroviral particle containing RNA encoding thepolypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide, or an VEGF-2 antibody, in vivo, for example, by proceduresknown in the art. As known in the art, a producer cell for producing aretroviral particle containing RNA encoding VEGF-2 antibodies of thepresent invention may be administered to a patient for engineering cellsin vivo and expression of VEGF-2 antibodies in vivo. These and othermethods for administering VEGF-2 antibodies of the present invention bysuch methods should be apparent to those skilled in the art from theteachings of the present invention. For example, the expression vehiclefor engineering cells may be other than a retroviral particle, forexample, an adenovirus, which may be used to engineer cells in vivoafter combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller et al., Biotechniques 7:980-990 (1989), or any other promoter(e.g., cellular promoters such as eukaryotic cellular promotersincluding, but not limited to, the histone, pol III, and b-actinpromoters). Other viral promoters which may be employed include, but arenot limited to, adenovirus promoters, thymidine kinase (TK) promoters,and B19 parvovirus promoters. The selection of a suitable promoter willbe apparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, y-2,y-AM, PA12, T19-14×, VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Diagnostic Assays

This invention is also related to the use of the VEGF-2 gene as part ofa diagnostic assay for detecting diseases or susceptibility to diseasesrelated to the presence of mutations in VEGF-2 nucleic acid sequences.

Individuals carrying mutations in the VEGF-2 gene may be detected at theDNA level by a variety of techniques. Nucleic acids for diagnosis may beobtained from a patient's cells, such as from blood, urine, saliva,tissue biopsy and autopsy material. The genomic DNA may be used directlyfor detection or may be amplified enzymatically by using PCR (Saiki etal., Nature 324:163-166 (1986)) prior to analysis. RNA or cDNA may alsobe used for the same purpose. As an example, PCR primers complementaryto the nucleic acid encoding VEGF-2 can be used to identify and analyzeVEGF-2 mutations. For example, deletions and insertions can be detectedby a change in size of the amplified product in comparison to the normalgenotype. Point mutations can be identified by hybridizing amplified DNAto radiolabeled VEGF-2 RNA or alternatively, radiolabeled VEGF-2antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of VEGF-2 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, abnormal cellular differentiation. Assays used todetect levels of VEGF-2 protein in a sample derived from a host arewell-known to those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays and“sandwich” assay. An ELISA assay (Coligan et al., Current Protocols inImmunology 1(2), Chapter 6, (1991)) initially comprises preparing anantibody specific to the VEGF-2 antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or, in this example, ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein, such as, bovineserum albumen. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any VEGF-2proteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to VEGF-2. Unattachedreporter antibody is then washed out. Peroxidase substrates are thenadded to the dish and the amount of color developed in a given timeperiod is a measurement of the amount of VEGF-2 protein present in agiven volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific toVEGF-2 are attached to a solid support. Polypeptides of the presentinvention are then labeled, for example, by radioactivity, and a samplederived from the host are passed over the solid support and the amountof label detected, for example by liquid scintillation chromatography,can be correlated to a quantity of VEGF-2 in the sample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayVEGF-2 is passed over a solid support and binds to antibody attached toa solid support. A second antibody is then bound to the VEGF-2. A thirdantibody which is labeled and specific to the second antibody is thenpassed over the solid support and binds to the second antibody and anamount can then be quantified.

Chromosome Identification

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphism's) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with probes from the cDNA asshort as 50 or 60 base pairs. For a review of this technique, see Vermaet al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that cDNA sequence. Ultimately, completesequencing of genes from several individuals is required to confirm thepresence of a mutation and to distinguish mutations from polymorphisms.

Antisense

The present invention is further directed to inhibiting VEGF-2 in vivoby the use of antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the maturepolynucleotide sequence, which encodes for the polypeptide of thepresent invention, is used to design an antisense RNA oligonucleotide offrom 10 to 40 base pairs in length. A DNA oligonucleotide is designed tobe complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney etal., Science 241:456 (1988); and Dervan et al. Science, 251:1360 (1991),thereby preventing transcription and the production of VEGF-2. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of an mRNA molecule into the VEGF-2 (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

Alternatively, the oligonucleotides described above can be delivered tocells by procedures in the art such that the anti-sense RNA or DNA maybe expressed in vivo to inhibit production of VEGF-2 in the mannerdescribed above.

Antisense constructs to VEGF-2, therefore, may inhibit the angiogenicactivity of the VEGF-2 and prevent the further growth or even regresssolid tumors, since angiogenesis and neovascularization are essentialsteps in solid tumor growth. These antisense constructs may also be usedto treat rheumatoid arthritis, psoriasis, diabetic retinopathy andKaposi's sarcoma which are all characterized by abnormal angiogenesis.

Epitope-Bearing Portions

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO: 18 (orfragments or variants thereof), or the full length polypeptide (orfragments or variant thereof), encoded by a polynucleotide sequencecontained in ATCC deposit Nos. 97149 or 75698 or encoded by apolynucleotide that hybridizes to the complement of the sequence of SEQID NOs: 1 or 3 or contained in ATCC deposit Nos. 97149 or 75698 understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNOs:2, 4, and 18), polynucleotide sequences of the complementary strandof a polynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the polypeptides of the presentinvention as well as polynucleotides encoding these epitopes-bearingportions. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 81:3998-4002.However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al. (1995) Mol. Gen. Genet.249:425-431. Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

It is particularly pointed out that the immunogenic epitopes comprisespredicted critical amino acid residues determined by the Jameson-Wolfanalysis. Thus, additional flanking residues on either the N-terminal,C-terminal, or both N- and C-terminal ends may be added to thesesequences to generate an epitope-bearing polypeptide of the presentinvention. Therefore, the immunogenic epitopes may include additionalN-terminal or C-terminal amino acid residues. The additional flankingamino acid residues may be contiguous flanking N-terminal and/orC-terminal sequences from the polypeptides of the present invention,heterologous polypeptide sequences, or may include both contiguousflanking sequences from the polypeptides of the present invention andheterologous polypeptide sequences.

Antibodies are preferably prepared from these regions or from discretefragments in these regions. However, antibodies can be prepared from anyregion of the peptide as described herein. A preferred fragment producesan antibody that diminishes or completely prevents binding of VEGF-2 toits receptor (e.g., flk-1, or flt-4). Antibodies can be developedagainst the full length VEGF-2 or portions of the receptor, for example,the secreted form of VEGF-2 polypeptide or any portions of theseregions. Antibodies may also be developed against specific functionalsites, such as the site of receptor binding or sites that areglycosylated, phosphorylated, myristoylated, or amidated.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

The immunogenic and antigenic epitope-bearing fragments may be specifiedby either the number of contiguous amino acid residues, as describedabove, or further specified by N-terminal and C-terminal positions ofthese fragments on the amino acid sequence of SEQ ID NO:2. Everycombination of a N-terminal and C-terminal position that a fragment of,for example, at least 7 or at least 15 contiguous amino acid residues inlength could occupy on the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:18 is included in the invention. Again, “at least 7contiguous amino acid residues in length” means 7 amino acid residues inlength or any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptide of the present invention.Specifically, each and every integer between 7 and the number of aminoacid residues of the full length polypeptide are included in the presentinvention.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

Preferred antigenic epitopes include the antigenic epitopes disclosedherein, as well as any combination of two, three, four, five or more ofthese antigenic epitopes. Antigenic epitopes can be used as the targetmolecules in immunoassays. (See, for instance, Wilson et al., Cell37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Preferred immunogenic epitopes include the immunogenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these immunogenic epitopes. The polypeptides comprising oneor more immunogenic epitopes may be presented for eliciting an antibodyresponse together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs), first described by J. P. Tam in Proc.Natl. Acad. Sci. U.S.A. 85:5409 which is incorporated by referenceherein in its entirety. MAPs consist of multiple copies of a specificpeptide attached to a non-immunogenic lysine core. Map peptides usuallycontain four or eight copies of the peptide often referred to as MAP-4or MAP-8 peptides. By way of non-limiting example, MAPs may besynthesized onto a lysine core matrix attached to a polyethyleneglycol-polystyrene (PEG-PS) support. The peptide of interest issynthesized onto the lysine residues using 9-fluorenylmethoxycarbonyl(Fmoc) chemistry. For example, Applied Biosystems (Foster City, Calif.)offers MAP resins, such as, for example, the Fmoc Resin 4 Branch and theFmoc Resin 8 Branch which can be used to synthesize MAPs. Cleavage ofMAPs from the resin is performed with standard trifloroacetic acid(TFA)-based cocktails known in the art. Purification of MAPs, except fordesalting, is not necessary. MAP peptides may be used as an immunizingvaccine which elicits antibodies that recognize both the MAP and thenative protein from which the peptide was derived.

Epitope bearing polypeptides of the invention may be modified, forexample, by the addition of amino acids at the amino- and/orcarboxy-termini of the peptide. Such modifications may be performed, forexample, to alter the conformation of the epitope bearing polypeptidesuch that the epitope will have a conformation more closely related tothe structure of the epitope in the native protein. An example of amodified epitope-bearing polypeptide of the invention is a polypeptidein which one or more cysteine residues have been added to thepolypeptide to allow for the formation of a disulfide bond between twocysteines, resulting in a stable loop structure of the epitope bearingpolypeptide under non-reducing conditions. Disulfide bonds may formbetween a cysteine residue added to the polypeptide and a cysteineresidue of the naturally occurring epitope, or may form between twocysteines which have both been added to the naturally occurring epitopebearing polypeptide. Additionally, it is possible to modify one or moreamino acid residues of the naturally occurring epitope bearingpolypeptide by substituting them with cysteines to promote the formationof disulfide bonded loop structures. Cyclic thioether molecules ofsynthetic peptides may be routinely generated using techniques known inthe art and are described in PCT publication WO 97/46251, incorporatedin its entirety by reference herein. Other modifications ofepitope-bearing polypeptides contemplated by this invention includebiotinylation.

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If in vivoimmunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides or MAP peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μgs of peptide or carrier protein and Freund'sadjuvant. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody which can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acidsI-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-x of human serum albumin, where x is an integerfrom 1 to 585 and the albumin fragment has human serum albumin activity.In another preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins as those described above may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 or SEQ ID NO:3 and the polypeptides encoded by thesepolynucleotides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments by homologous or site-specificrecombination to generate variation in the polynucleotide sequence. Inanother embodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. polypeptide, polypeptide fragment, or variant of SEQ ID NO:2,SEQ ID NO:4 OR SEQ ID NO:18, or the full length polypeptide (orfragments or variant thereof), the pro-protein polypeptide sequence, orthe secreted polypeptide encoded by a polynucleotide sequence containedin ATCC deposit Nos. 97149 or 75698, and/or an epitope, of the presentinvention (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding).

The VEGF-2 polypeptides bound by the antibodies of the invention may bein monomers or multimers (i.e., dimers, trimers, tetramers and highermultimers). Accordingly, the present invention relates to antibodiesthat bind monomers and multimers of the VEGF-2 polypeptides of theinvention, their preparation, and compositions (preferably,pharmaceutical compositions) containing them. In specific embodiments,the VEGF-2 polypeptides bound by the antibodies of the invention aremonomers, dimers, trimers or tetramers. In additional embodiments, thepolypeptides bound by the antibodies of the invention of the inventionare at least dimers, at least trimers, or at least tetramers.

Multimeric VEGF-2 bound by the antibodies of the invention may behomomers or heteromers. A VEGF-2 homomer, refers to a multimercontaining only VEGF-2 polypeptides (including VEGF-2 fragments,variants, and fusion proteins, as described herein). These homomers maycontain VEGF-2 polypeptides having identical or different amino acidsequences. In specific embodiments, the VEGF-2 multimer bound byantibodies of the invention is a homodimer (e.g., containing two VEGF-2polypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing three VEGF-2 polypeptides having identicalor different amino acid sequences). In a preferred embodiment, theantibodies of the invention bind homotrimers of VEGF-2. In additionalembodiments, the homomeric VEGF-2 multimer bound by the antibodies ofthe invention is at least a homodimer, at least a homotrimer, or atleast a homotetramer.

Heteromeric VEGF-2 refers to a multimer containing heterologouspolypeptides (i.e., polypeptides of a different protein) in addition tothe VEGF-2 polypeptides of the invention. In a specific embodiment, theVEGF-2 multimer bound by the antibodies of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the heteromeric VEGF-2 multimer bound by the antibodies ofthe invention is at least a heterodimer, at least a heterotrimer, or atleast a heterotetramer.

VEGF-2 multimers bound by the antibodies of the invention may be theresult of hydrophobic, hydrophilic, ionic and/or covalent associationsand/or may be indirectly linked, by for example, liposome formation.Thus, in one embodiment, VEGF-2 multimers, such as, for example,homodimers or homotrimers, are formed when polypeptides of the inventioncontact one another in solution. In another embodiment, VEGF-2heteromultimers, such as, for example, VEGF-2 heterotrimers or VEGF-2heterotetramers, are formed when polypeptides of the invention contactantibodies to the polypeptides of the invention (including antibodies tothe heterologous polypeptide sequence in a fusion protein of theinvention) in solution. In other embodiments, VEGF-2 multimers areformed by covalent associations with and/or between the VEGF-2polypeptides of the invention. Such covalent associations may involveone or more amino acid residues contained in the polypeptide sequence(e.g., that recited in SEQ ID NO:2, SEQ ID NO:14 or SEQ ID NO:18). Inone instance, the covalent associations are cross-linking betweencysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a VEGF-2 fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In aspecific example, the covalent associations are between the heterologoussequence contained in a VEGF-2-Fc fusion protein. In another specificexample, covalent associations of fusion proteins of the invention arebetween heterologous polypeptide sequence from another PDGF/VEGF familyligand/receptor member that is capable of forming covalently associatedmultimers. Examples include those peptide linkers described in U.S. Pat.No. 5,073,627 (hereby incorporated by reference). Proteins comprisingmultiple VEGF-2 polypeptides separated by peptide linkers may beproduced using conventional recombinant DNA technology.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody binding site.

Thus, an intact IgG antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe heavy and the light chains of each pair are aligned by the frameworkregions, enabling binding to a specific epitope. From N-terminal toC-terminal, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547 1553 (1992). In addition, bispecificantibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’:small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448(1993)) or “Janusins” (Traunecker et al. “Bispecific single chainmolecules (Janusins) target cytotoxic lymphocytes on HIV infected cells”EMBO J. 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992)).

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), intracellularly-made antibodies (i.e., intrabodies), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The antibodies of the present invention include IgG (includingIgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, orIgM, and IgY. In a preferred embodiment, the immunoglobulin is an IgG1isotype. In another preferred embodiment, the immunoglobulin is an IgG2isotype. In another preferred embodiment, the immunoglobulin is an IgG4isotype. Immunoglobulins may have both a heavy and light chain. An arrayof IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with alight chain of the kappa or lambda forms.

Most preferably the antibodies are human antigen binding antibodyfragments of the present invention include, but are not limited to, Fab,Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. The antibodies may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, rabbit,goat, guinea pig, camel, horse, or chicken. Antigen-binding antibodyfragments, including single-chain antibodies, may comprise the variableregion(s) alone or in combination with the entirety or a portion of thefollowing: hinge region, CH1, CH2, and CH3 domains. Also included in theinvention are antigen-binding fragments also comprising any combinationof variable region(s) with a hinge region, CH1, CH2, and CH3 domains.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodieswhich are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures. Inpreferred embodiments, the antibodies of the invention bind the fulllength VEGF-2 protein encoded by a polynucleotide sequence contained inATCC deposit Nos. 97149 or 75698. In preferred embodiments, theantibodies of the invention bind the pro-protein form of the VEGF-2protein encoded by a polynucleotide sequence contained in ATCC depositNos. 97149 or 75698. In preferred embodiments, the antibodies of theinvention bind the secreted VEGF-2 protein encoded by a polynucleotidesequence contained in ATCC deposit Nos. 97149 or 75698. In otherpreferred embodiments, the antibodies of the invention bind the secretedVEGF-2 protein but not the full length VEGF-2 protein encoded by apolynucleotide sequence contained in ATCC deposit Nos. 97149 or 75698.In other preferred embodiments, the antibodies of the invention bindboth the secreted form of VEGF-2 protein and the full length VEGF-2protein encoded by a polynucleotide sequence contained in ATCC depositNos. 97149 or 75698.

In other preferred embodiments, the antibodies of the invention bindamino acids 103 to 227 of SEQ ID NO: 18. In other embodiments theantibodies of the invention bind dimeric VEGF-2 polypeptides consistingof two polypeptides each consisting of amino acids 103 to 227 of SEQ IDNO:18. In still other preferred embodiments, the antibodies of theinvention bind amino acids 112 to 227 of SEQ ID NO:18. In still otherembodiments the antibodies of the invention bind dimeric VEGF-2polypeptides consisting of two polypeptides each consisting of aminoacids 112 to 227 of SEQ ID NO: 18.

Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. In specific embodiments, antibodiesof the present invention cross-react with murine, monkey, rat and/orrabbit homologs of human proteins and the corresponding epitopesthereof. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In a specific embodiment, the above-describedcross-reactivity is with respect to any single specific antigenic orimmunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of thespecific antigenic and/or immunogenic polypeptides disclosed herein.Further included in the present invention are antibodies which only bindpolypeptides encoded by polynucleotides which hybridize to apolynucleotide of the present invention under stringent hybridizationconditions (as described herein).

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof)immunospecifically bind to a polypeptide or polypeptide fragment orvariant of human VEGF-2 (SEQ ID NO:2, SEQ ID NO:4 OR SEQ ID NO:18)and/or monkey VEGF-2. Preferably, the antibodies of the invention bindimmunospecifically to human VEGF-2. Preferably, the antibodies of theinvention bind immunospecifically to human and monkey VEGF-2. Alsopreferably, the antibodies of the invention bind immunospecifically tohuman VEGF-2 and murine VEGF-2. More preferably, antibodies of theinvention, bind immunospecifically and with higher affinity to humanVEGF-2 than to murine VEGF-2.

In preferred embodiments, the antibodies of the present invention(including molecules comprising, or alternatively consisting of,antibody fragments or variants thereof), immunospecifically bind toVEGF-2 and do not cross-react with any other antigens. In preferredembodiments, the antibodies of the invention immunspecifically bind toVEGF-2 and do not cross-react with other members of the VEGF/PDGF familysuch as, for example, VEGF, VEGF-1, VEGF-3 (VEGF-B), VEGF-4 (VEGF-D),PDGFa or PDGFb.

In other preferred embodiments, the antibodies of the inventionimmunspecifically bind to VEGF-2 and cross-react with other members ofthe VEGF/PDGF family such as, for example, VEGF, VEGF-1, VEGF-3(VEGF-B), VEGF-4 (VEGF-D), PDGFa or PDGFb.

In a preferred embodiment, antibodies of the invention preferentiallybind VEGF-2 (SEQ ID NO:2, SEQ ID NO:4 OR SEQ ID NO:18), or fragments andvariants thereof relative to their ability to bind other antigens, (suchas, for example, other chemokine receptors).

By way of non-limiting example, an antibody may be considered to bind afirst antigen preferentially if it binds said first antigen with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second antigen. In another non-limiting embodiment, an antibody maybe considered to bind a first antigen preferentially if it binds saidfirst antigen with an affinity that is at least one order of magnitudeless than the antibody's K_(D) for the second antigen. In anothernon-limiting embodiment, an antibody may be considered to bind a firstantigen preferentially if it binds said first antigen with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second antigen.

In another non-limiting embodiment, an antibody may be considered tobind a first antigen preferentially if it binds said first antigen withan off rate (k_(off)) that is less than the antibody's k_(off) for thesecond antigen. In another non-limiting embodiment, an antibody may beconsidered to bind a first antigen preferentially if it binds said firstantigen with an affinity that is at least one order of magnitude lessthan the antibody's k_(off) for the second antigen. In anothernon-limiting embodiment, an antibody may be considered to bind a firstantigen preferentially if it binds said first antigen with an affinitythat is at least two orders of magnitude less than the antibody'sk_(off) for the second antigen.

Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M.More preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M,10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M. Even more preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included are neutralizing antibodies which bind the ligand andprevent binding of the ligand to the receptor, as well as antibodieswhich bind the ligand, thereby preventing receptor activation, but donot prevent the ligand from binding the receptor. Further included areantibodies which activate the receptor. These antibodies may act asagonists for either all or less than all of the biological activitiesaffected by ligand-mediated receptor activation. The antibodies may bespecified as agonists or antagonists for biological activitiescomprising specific activities disclosed herein. The above antibodyagonists can be made using methods known in the art. See e.g., WO96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al. (1998) Blood92(6):1981-1988; Chen, Z. et al. (1998) Cancer Res. 58(16):3668-3678;Harrop, J. A. et al. (1998) J. Immunol. 161(4):1786-1794; Zhu, Z. et al.(1998) Cancer Res. 58(15):3209-3214; Yoon, D. Y. et al. (1998) J.Immunol. 160(7):3170-3179; Prat, M. et al. (1998) J. Cell. Sci. 111(Pt2):237-247; Pitard, V. et al. (1997) J. Immunol. Methods 205(2):177-190;Liautard, J. et al. (1997) Cytokinde 9(4):233-241; Carlson, N. G. et al.(1997) J. Biol. Chem. 272(17):11295-11301; Taryman, R. E. et al. (1995)Neuron 14(4):755-762; Muller, Y. A. et al. (1998) Structure6(9):1153-1167; Bartunek, P. et al. (1996) Cytokine 8(1): 14-20 (saidreferences incorporated by reference in their entireties).

The invention also encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that have one or more of the same biologicalcharacteristics as one or more of the antibodies described herein. By“biological characteristics” is meant, the in vitro or in vivoactivities or properties of the antibodies, such as, for example, theability to inhibit VEGF-2 binding to its receptor (e.g., flk-1 and/orflt-4) (e.g., see Example 33), the ability to inhibit VEGF-2 inducedphosphorylation of Elk-1 (e.g., see Example 35), the ability to inhibitVEGF-2 induced proliferation of vascular and/or lymphatic endothelialcells (e.g., see Example 34), the ability to inhibit angiogenesis (e.g.,see Examples 16 and 23), and/or the ability to inhibit tumor growthand/or tumor metastasis (e.g., see Examples 37 and 38). Optionally, theantibodies of the invention will bind to the same epitope as at leastone of the antibodies specifically referred to herein. Such epitopebinding can be routinely determined using assays known in the art.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that neutralize VEGF-2, said antibodies comprising,or alternatively consisting of, a portion (e.g., VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2, and/or VL CDR3) of a VH or VL domain of an scFvreferred to in Table 2. An antibody that “neutralizes VEGF-2 or afragment or variant thereof” is, for example, an antibody that inhibitsVEGF-2 binding to its receptor (e.g., flk-1 and/or flt-4) (e.g., seeExample 33), inhibits VEGF-2 induced phosphorylation of Elk-1 (e.g., seeExample 35), inhibits VEGF-2 induced proliferation of vascular and/orlymphatic endothelial cells (e.g., see Example 34), inhibitsangiogenesis (e.g., see Examples 16 and 23), and/or inhibits tumorgrowth and/or tumor metastasis (e.g., see Examples 37 and 38). In oneembodiment, an antibody that neutralizes VEGF-2, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH domain of an scFv referred to in Table 2, or a fragment orvariant thereof and a VL domain of an scFv referred to in Table 2, or afragment or variant thereof. In another embodiment, an antibody thatneutralizes VEGF-2, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain and a VLdomain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that neutralizes VEGF-2, comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH domain of anscFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that neutralizes VEGF-2, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL domain of an scFv referred to in Table 2, or a fragment orvariant thereof. In another embodiment, an antibody that neutralizesVEGF-2 or a fragment or variant thereof, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH CDRdomain of an scFv referred to in Table 2, or a fragment or variantthereof. In a preferred embodiment, an antibody that neutralizes VEGF-2or a fragment or variant thereof, comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. In anotherembodiment, an antibody that neutralizes VEGF-2 or a fragment or variantthereof, comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VL CDR of an scFv referred to in Table 2,or a fragment or variant thereof. In another preferred embodiment, anantibody that neutralizes VEGF-2 or a fragment or variant thereof,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VL CDR3 of an scFv referred to in Table 2, or afragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibits VEGF-2 binding to its receptor (e.g.,flk-1 and/or flt-4) (e.g. see Example 33). Said antibodies may comprise,or alternatively consist of, a portion (e.g., VH CDR1, VH CDR2, VH CDR3,VL CDR1, VL CDR2, or VL CDR3) of a VH or VL domain having an amino acidsequence of an scFv referred to in Table 2 or a fragment or variantthereof. In one embodiment, an antibody that inhibits VEGF-2 binding toits receptor (e.g., flk-1 and/or flt-4) comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainof an scFv referred to in Table 2, or a fragment or variant thereof anda VL domain of an scFv referred to in Table 2, or a fragment or variantthereof. In another embodiment, an antibody that inhibits VEGF-2 bindingto its receptor (e.g., flk-1 and/or flt-4) comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainand a VL domain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that inhibits VEGF-2 binding to its receptor (e.g., flk-1and/or flt-4) comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VH domain of an scFv referred to inTable 2, or a fragment or variant thereof. In another embodiment, anantibody that inhibits VEGF-2 binding to its receptor (e.g., flk-1and/or flt-4) comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VL domain of an scFv referred to inTable 2, or a fragment or variant thereof. In a preferred embodiment, anantibody that inhibits VEGF-2 binding to its receptor (e.g., flk-1and/or flt-4) comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VH CDR3 of an scFv referred to inTable 2, or a fragment or variant thereof. In another preferredembodiment, an antibody that inhibits VEGF-2 binding to its receptor(e.g., flk-1 and/or flt-4) comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VL CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit VEGF-2 induced phosphorylation of Elk-1as determined by any method known in the art such as, for example, theassays described in Example 35. Said antibodies may comprise, oralternatively consist of, a portion (e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, or VL CDR3) of a VH or VL domain having an amino acidsequence of an scFv referred to in Table 2 or a fragment or variantthereof. In one embodiment, an antibody that inhibits VEGF-2 inducedphosphorylation of Elk-1, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain of an scFvreferred to in Table 2, or a fragment or variant thereof and a VL domainof an scFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that inhibits VEGF-2 inducedphosphorylation of Elk-1, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain and a VLdomain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that inhibits VEGF-2 induced phosphorylation of Elk-1,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain of an scFv referred to in Table 2, or afragment or variant thereof. In another embodiment, an antibody thatinhibits VEGF-2 induced phosphorylation of Elk-1, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL domain of an scFv referred to in Table 2, or a fragment orvariant thereof. In a preferred embodiment, an antibody that inhibitsVEGF-2 induced phosphorylation of Elk-1, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH CDR3of an scFv referred to in Table 2, or a fragment or variant thereof. Inanother preferred embodiment, an antibody inhibits VEGF-2 inducedphosphorylation of Elk-1, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VL CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that inhibit VEGF-2 induced proliferation of vascularand/or lymphatic endothelial cells (e.g., see Example 34). Saidantibodies may comprise, or alternatively consist of, a portion (e.g.,VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of a VH or VLdomain having an amino acid sequence of an scFv referred to in Table 2or a fragment or variant thereof. In one embodiment, an antibody thatinhibits VEGF-2 induced proliferation of vascular and/or lymphaticendothelial cells, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain of an scFvreferred to in Table 2, or a fragment or variant thereof and a VL domainof an scFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that inhibits VEGF-2 inducedproliferation of vascular and/or lymphatic endothelial cells, comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a VH domain and a VL domain from a single antibody (or scFvor Fab fragment) of the invention, or fragments or variants thereof. Inone embodiment, an antibody that inhibits VEGF-2 induced proliferationof vascular and/or lymphatic endothelial cells, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH domain of an scFv referred to in Table 2, or a fragment orvariant thereof. In another embodiment, an antibody that inhibits VEGF-2induced proliferation of vascular and/or lymphatic endothelial cells,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VL domain of an scFv referred to in Table 2, or afragment or variant thereof. In a preferred embodiment, an antibody thatinhibits VEGF-2 induced proliferation of vascular and/or lymphaticendothelial cells, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. In anotherpreferred embodiment, an antibody that inhibits VEGF-2 inducedproliferation of vascular and/or lymphatic endothelial cells, comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a VL CDR3 of an scFv referred to in Table 2, or a fragmentor variant thereof. Nucleic acid molecules encoding these antibodies arealso encompassed by the invention.

In highly preferred embodiments, the present invention also provides forantibodies (including molecules comprising, or alternatively consistingof, antibody fragments or variants thereof), that inhibit angiogenesis(e.g., see Examples 16 and 24). Said antibodies may comprise, oralternatively consist of, a portion (e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, or VL CDR3) of a VH or VL domain having an amino acidsequence of an scFv referred to in Table 2 or a fragment or variantthereof. In one embodiment, an antibody that inhibits angiogenesiscomprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain of an scFv referred to in Table 2, or afragment or variant thereof and a VL domain of an scFv referred to inTable 2, or a fragment or variant thereof. In another embodiment, anantibody that inhibits angiogenesis comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH domain and a VLdomain from a single antibody (or scFv or Fab fragment) of theinvention, or fragments or variants thereof. In one embodiment, anantibody that inhibits angiogenesis comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VH domain of anscFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that inhibits angiogenesis comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL domain of an scFv referred to in Table 2, or a fragment orvariant thereof. In a preferred embodiment, an antibody that inhibitsangiogenesis comprises, or alternatively consists of, a polypeptidehaving the amino acid sequence of a VH CDR3 of an scFv referred to inTable 2, or a fragment or variant thereof. In another preferredembodiment, an antibody that inhibits angiogenesis comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL CDR3 of an scFv referred to in Table 2, or a fragment or variantthereof. Nucleic acid molecules encoding these antibodies are alsoencompassed by the invention.

In other highly preferred embodiments, the present invention alsoprovides for antibodies (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),that inhibit tumor growth and/or tumor metastasis (e.g., see Examples 37and 38). Said antibodies may comprise, or alternatively consist of, aportion (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3)of a VH or VL domain having an amino acid sequence of an scFv referredto in Table 2 or a fragment or variant thereof. In one embodiment, anantibody that inhibits tumor growth and/or tumor metastasis comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a VH domain of an scFv referred to in Table 2, or a fragmentor variant thereof and a VL domain of an scFv referred to in Table 2, ora fragment or variant thereof. In another embodiment, an antibody thatinhibits tumor growth and/or tumor metastasis comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH domain and a VL domain from a single antibody (or scFv or Fabfragment) of the invention, or fragments or variants thereof. In oneembodiment, an antibody that inhibits tumor growth and/or tumormetastasis comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of a VH domain of an scFv referred to in Table2, or a fragment or variant thereof. In another embodiment, an antibodythat inhibits tumor growth and/or tumor metastasis comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VL domain of an scFv referred to in Table 2, or a fragment orvariant thereof. In a preferred embodiment, an antibody that inhibitstumor growth and/or tumor metastasis comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH CDR3of an scFv referred to in Table 2, or a fragment or variant thereof. Inanother preferred embodiment, an antibody that inhibits tumor growthand/or tumor metastasis comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VL CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that enhance the activity of VEGF-2, said antibodiescomprising, or alternatively consisting of, a portion (e.g., VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, or VL CDR3) of a VH or VL domain of anscFv referred to in Table 2, or a fragment or variant thereof. By way ofnon-limiting example, an antibody that “enhances the activity of VEGF-2or a fragment or variant thereof” is an antibody increases the abilityof VEGF-2 to bind to its receptor (e.g., flk-1 or flt-4), to stimulatethe VEGF-2 signalling cascade (e.g., increase VEGF-2 inducedphosphorylation of Elk-1, See Example 35), to induce proliferation ofvascular and/or lymphatic endothelial cells (e.g. see Example 34), andor to promote angiogenesis. In one embodiment, an antibody that enhancesthe activity of VEGF-2, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH domain of an scFvreferred to in Table 2, or a fragment or variant thereof and a VL domainof an scFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that enhances the activity of VEGF-2,comprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a VH domain and a VL domain from a single antibody (orscFv or Fab fragment) of the invention, or fragments or variantsthereof. In one embodiment, an antibody that enhances the activity ofVEGF-2 or a fragment or variant thereof, comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a VH domainof an scFv referred to in Table 2, or a fragment or variant thereof. Inanother embodiment, an antibody that enhances the activity of VEGF-2 ora fragment or variant thereof, comprises, or alternatively consists of,a polypeptide having the amino acid sequence of a VL domain of an scFvreferred to in Table 2, or a fragment or variant thereof. In anotherembodiment, an antibody that enhances the activity of VEGF-2 or afragment or variant thereof, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VH CDR domain referredto in of an scFv referred to in Table 2 or a fragment or variantthereof. In a preferred embodiment, an antibody that enhances theactivity of VEGF-2 or a fragment or variant thereof, comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a VH CDR3 of an scFv referred to in Table 2, or a fragment or variantthereof. In another embodiment, an antibody that enhances VEGF-2 or afragment or variant thereof, comprises, or alternatively consists of, apolypeptide having the amino acid sequence of a VL CDR domain of an scFvreferred to in Table 2, or a fragment or variant thereof. In anotherpreferred embodiment, an antibody that enhances the activity of VEGF-2or a fragment or variant thereof, comprises, or alternatively consistsof, a polypeptide having the amino acid sequence of a VL CDR3 of an scFvreferred to in Table 2, or a fragment or variant thereof. Nucleic acidmolecules encoding these antibodies are also encompassed by theinvention.

The present invention also provides for fusion proteins comprising, oralternatively consisting of, an antibody (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that immunospecifically binds to VEGF-2, and aheterologous polypeptide. Preferably, the heterologous polypeptide towhich the antibody is fused to is useful for function or is useful totarget the VEGF-2 antibodies to specific cells (tumor cells, especiallyprostate, breast, brain or colon cancer cells). In one embodiment, afusion protein of the invention comprises, or alternatively consists of,a polypeptide having the amino acid sequence of any one or more of theVH domains of an scFv referred to in Table 2 or the amino acid sequenceof any one or more of the VL domains of an scFv referred to in Table 2or fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein of the presentinvention comprises, or alternatively consists of, a polypeptide havingthe amino acid sequence of any one, two, three, or more of the VH CDRsof an scFv referred to in Table 2, or the amino acid sequence of anyone, two, three, or more of the VL CDRs of an scFv referred to in Table2, or fragments or variants thereof, and a heterologous polypeptidesequence. In a preferred embodiment, the fusion protein comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof, a VH CDR3 of an scFv referred to in Table 2, or fragments or variantthereof, and a heterologous polypeptide sequence, which fusion proteinimmunospecifically binds to VEGF-2. In another embodiment, a fusionprotein comprises, or alternatively consists of a polypeptide having theamino acid sequence of at least one VH domain of an scFv referred to inTable 2 and the amino acid sequence of at least one VL domain of an scFvreferred to in Table 2 or fragments or variants thereof, and aheterologous polypeptide sequence. Preferably, the VH and VL domains ofthe fusion protein correspond to a single antibody (or scFv or Fabfragment) of the invention. In yet another embodiment, a fusion proteinof the invention comprises, or alternatively consists of a polypeptidehaving the amino acid sequence of any one, two, three or more of the VHCDRs of an scFv referred to in Table 2 and the amino acid sequence ofany one, two, three or more of the VL CDRs of an scFv referred to inTable 2, or fragments or variants thereof, and a heterologouspolypeptide sequence. Preferably, two, three, four, five, six, or moreof the VHCDR(s) or VLCDR(s) correspond to single antibody (or scFv orFab fragment) of the invention. Nucleic acid molecules encoding thesefusion proteins are also encompassed by the invention.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

By way of another non-limiting example, antibodies of the invention maybe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention may be used forepitope mapping to identify the epitope(s) bound by the antibody.Epitopes identified in this way may, in turn, for example, be used asvaccine candidates, i.e., to immunize an individual to elicit antibodiesagainst the naturally occurring forms of VEGF-2.

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide of techniques knownin the art including the use of hybridoma and recombinant technology.See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in:MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y.,1981) (said references incorporated by reference in their entireties).The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP2/0 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Another well known method for producing both polyclonal and monoclonalhuman B cell lines is transformation using Epstein Barr Virus (EBV).Protocols for generating EBV-transformed B cell lines are commonly knownin the art, such as, for example, the protocol outlined in Chapter 7.22of Current Protocols in Immunology, Coligan et al., Eds., 1994, JohnWiley & Sons, NY, which is hereby incorporated in its entirety byreference herein. The source of B cells for transformation is commonlyhuman peripheral blood, but B cells for transformation may also bederived from other sources including, but not limited to, lymph nodes,tonsil, spleen, tumor tissue, and infected tissues. Tissues aregenerally made into single cell suspensions prior to EBV transformation.Additionally, steps may be taken to either physically remove orinactivate T cells (e.g., by treatment with cyclosporin A) in Bcell-containing samples, because T cells from individuals seropositivefor anti-EBV antibodies can suppress B cell immortalization by EBV. Ingeneral, the sample containing human B cells is innoculated with EBV,and cultured for 3-4 weeks. A typical source of EBV is the culturesupernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs ofEBV transformation can generally be seen towards the end of the 3-4 weekculture period. By phase-contrast microscopy, transformed cells mayappear large, clear, hairy and tend to aggregate in tight clusters ofcells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human×mouse; e.g., SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al.(1995) J. Immunol. Methods 184:177-186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene 1879-18; Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864-869; and Sawai, H. et al. (1995) AJRI 34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988) Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entirety. Humanized antibodies are antibody molecules fromnon-human species antibody that binds the desired antigen having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework regions from a human immunoglobulin molecule.Often, framework residues in the human framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.)

Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805-814; Roguska M. A. et al. (1994)PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645(said references incorporated by reference in their entireties).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Fremont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby activate orblock its biological activity.

Intrabodies are antibodies, often scFvs, that expressed from arecombinant nucleic acid molecule and engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm). Intrabodies may be used, for example, to ablate thefunction of a protein to which the intrabody binds. The expression ofintrabodies may also be regulated through the use of inducible promotersin the nucleic acid expression vector comprising the intrabody.Intrabodies of the invention can be produced using methods known in theart, such as those disclosed and reviewed in Chen et al., Hum. GeneTher. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997);Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba etal., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz andSteipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods231:207-222 (1999); and references cited therein. In particular, a CCR5intrabody has been produced by Steinberger et al., Proc. Natl. Acad.Sci. USA 97:805-810 (2000).

XenoMouse Technology

Antibodies in accordance with the invention may be prepared theutilizing transgenic mouse that has a substantial portion of the humanantibody producing genome inserted but that is rendered deficient in theproduction of endogenous, murine, antibodies (e.g., XenoMouse strainsavailable from Abgenix Inc., Fremont, Calif.). Such mice, then, arecapable of producing human immunoglobulin molecules and antibodies andare deficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilized for achieving the same are disclosedin the patents, applications, and references disclosed herein.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs) an important milestone towards fulfilling the promiseof antibody therapy in human disease.

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized Monoclonalantibodies and thus to increase the efficacy and safety of theadministered antibodies. The use of fully human antibodies can beexpected to provide a substantial advantage in the treatment of chronicand recurring human diseases, such as cancer, which require repeatedantibody administrations.

One approach towards this goal was to engineer mouse strains deficientin mouse antibody production with large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human Monoclonalantibodies with the desired specificity could be readily produced andselected.

This general strategy was demonstrated in connection with the generationof the first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). The XenoMouse™ strains were engineeredwith yeast artificial chromosomes (YACS) containing 245 kb and 10 190kb-sized germline configuration fragments of the human heavy chain locusand kappa light chain locus, respectively, which contained core variableand constant region sequences. Id. The human Ig containing YACs provedto be compatible with the mouse system for both rearrangement andexpression of antibodies and were capable of substituting for theinactivated mouse Ig genes. This was demonstrated by their ability toinduce B-cell development, to produce an adult-like human repertoire offully human antibodies, and to generate antigen-specific humanmonoclonal antibodies. These results also suggested that introduction oflarger portions of the human Ig loci containing greater numbers of Vgenes, additional regulatory elements, and human Ig constant regionsmight recapitulate substantially the full repertoire that ischaracteristic of the human humoral response to infection andimmunization. The work of Green et al. was recently extended to theintroduction of greater than approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse™ mice. See Mendezet al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp.Med. 188:483-495 (1998), Green, Journal of Immunological Methods231:11-23 (1999) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996, the disclosures of which are hereby incorporated byreference.

Such approach is further discussed and delineated in U.S. patentapplication Ser. Nos. 07/466,008, filed Jan. 12, 1990, 07/710,515, filedNov. 8, 1990, 07/919,297, filed Jul. 24, 1992, 07/922,649, filed Jul.30, 1992, 08/031,801, filed Mar. 15, 1993, 08/112,848, filed Aug. 27,1993, 08/234,145, filed Apr. 28, 1994, 08/376,279, filed Jan. 20, 1995,08/430, 938, Apr. 27, 1995, 08/464,584, filed Jun. 5, 1995, 08/464,582,filed Jun. 5, 1995, 08/471,191, filed Jun. 5, 1995, 08/462,837, filedJun. 5, 1995, 08/486,853, filed Jun. 5, 1995, 08/486,857, filed Jun. 5,1995, 08/486,859, filed Jun. 5, 1995, 08/462,513, filed Jun. 5, 1995,08/724,752, filed Oct. 2, 1996, and 08/759,620, filed Dec. 3, 1996. Seealso Mendez et al. Nature Genetics 15:146-156 (1997) and Green andJakobovits J. Exp. Med. 188:483 495 (1998). See also European PatentNo., EP 0 471 151 B1, grant published Jun. 12, 1996, InternationalPatent Application No., WO 94/02602, published Feb. 3, 1994,International Patent Application No., WO 96/34096, published Oct. 31,1996, and WO 98/24893, published Jun. 11, 1998. The disclosures of eachof the above-cited patents, applications, and references are herebyincorporated by reference in their entirety.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against VEGF-2 polypeptides in order to vitiate concernsand/or effects of HAMA or HACA responses.

Using phage display technology, the present inventors have identifiedsingle chain antibody molecules (“scFvs”) that immunospecifically bindto VEGF-2. These scFvs are listed in Table 2. For the scFvs that havebeen deposited with the American Type Tissue Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209, the ATCC Deposit number anddate of deposit are also provided in Table 2. Molecules comprising, oralternatively consisting of, fragments or variants of these scFvs (e.g.,including VH domains, VH CDRs, VL domains, or VL CDRs having an aminoacid sequence of any one of those referenced to in Table 2), thatimmunospecifically bind to VEGF-2 (or fragments or variants thereof,including the pro-protein from of VEGF-2 and secreted form of VEGF-2)are also encompassed by the invention, as are nucleic acid moleculesthat encode these scFvs, and/or molecules.

In particular, the invention relates to scFvs comprising, oralternatively consisting of, an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 72-78 preferably SEQ ID NOs:72 and 73 asreferred to in Table 2 below. Molecules comprising, or alternativelyconsisting of, fragments or variants of these scFvs (e.g., including VHdomains, VH CDRs, VL domains, or VL CDRs having an amino acid sequenceof any one of those referred to in Table 2), that immunospecificallybind to VEGF-2 are also encompassed by the invention, as are nucleicacid molecules that encode these scFvs, and/or molecules.

The present invention provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that immunospecifically bind to a polypeptide or apolypeptide fragment of VEGF-2. In particular, the invention providesantibodies corresponding to the scFvs referred to in Table 2, such scFvsmay routinely be “converted” to immunoglobulin molecules by inserting,for example, the nucleotide sequences encoding the VH and/or VL domainsof the scFv into an expression vector containing the constant domainsequences and engineered to direct the expression of the immunoglobulinmolecule, as described in more detail in Example 32, below.

TABLE 2 scFvs that Immunospecifically bind to VEGF-2 scFv AAs of AAs ofAAs of AAs of AAs of AAs of AAs of AAs of Cell Line ATCC SEQ ID VH VH VHVH VL VL VL VL Expressing Deposit ATCC Deposit scFv NO: Domain CDR1 CDR2CDR3 Domain CDR1 CDR2 CDR3 antibody Number Date 69D09 79 1-118 26-3550-66 99-107 136-247 158-170 186-192 225-236 NSO- PTA-4095 Feb. 21, 2002Mouse Myeloma 72D09 80 1-118 26-35 50-66 99-107 135-246 157-169 185-191224-235 25A07 81 1-118 26-35 50-66 99-107 136-247 141-153 169-175208-219 NSO- PTA-4179 Mar. 25, 2002 Mouse Myeloma 32G10X 82 1-128 26-3550-66 99-117 129-235 151-161 177-183 216-224 NSO- PTA-4096 Feb. 21, 2002Mouse Myeloma 30E06X 83 1-128 26-35 50-66 99-117 129-235 151-161 177-183216-224 NSO- PTA-4180 Mar. 25, 2002 Mouse Myeloma 17DO6 72 1-122 26-3550-66 99-110 139-250 161-173 189-195 228-239 16C10 73 1-129 26-35 50-6699-117 146-252 168-178 194-200 233-241 16B06 74 1-122 26-35 50-66 99-110138-247 160-173 189-196 228-238 19B09 75 1-124 26-37 52-67 100-112 140-251 162-175 191-197 230-240 20D05 76 1-127 26-35 50-66 99-115143-253 165-177 193-199 232-242 20G02 77 1-118 26-35 50-66 99-106134-244 156-169 185-191 224-233 20G11 78 1-121 26-35 50-66 99-109138-248 160-172 188-194 229-237

In one embodiment, the present invention provides the scFv of SEQ IDNO:72 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:72 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:73 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:73 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:74 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:74 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:75 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:75 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:76 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:76 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:77 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:77 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:78 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:78 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ if)NO:79 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:79 (or fragments or variants thereof), for example, as foundin SEQ ID NO: 84.

In one embodiment, the present invention provides the scFv of SEQ IDNO:80 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:80 (or fragments or variants thereof), for example, as foundin SEQ ID NO: 85.

In one embodiment, the present invention provides the scFv of SEQ IDNO:81 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:81 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:82 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:82 (or fragments or variants thereof).

In one embodiment, the present invention provides the scFv of SEQ IDNO:83 (or fragments or variants thereof). In another embodiment, thepresent invention provides nucleic acid molecules encoding the scFv ofSEQ ID NO:83 (or fragments or variants thereof).

The present invention encompasses antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof) that immunospecifically bind to a VEGF-2 polypeptideor a fragment, variant, or fusion protein thereof. A VEGF-2 polypeptideincludes, but is not limited to, the VEGF-2 polypeptide of SEQ ID NO:2,SEQ ID NO:4 OR SEQ ID NO:18 or the polypeptide encoded by the cDNAcontained in ATCC Deposit Numbers 97149 or 75698 deposited May 12, 1995and May 4, 1995, respectively. The VEGF-2 polypeptide bound by theantibodies of the invention may be the full-length protein, thepro-protein, or the secreted form of VEGF-2. VEGF-2 may be producedthrough recombinant expression of nucleic acids encoding thepolypeptides of SEQ ID NO:2, SEQ ID NO:4 OR SEQ ID NO:18, (e.g., thecDNA in ATCC Deposit Numbers 97149 or 75698).

In one embodiment of the present invention, antibodies thatimmunospecifically bind to a VEGF-2 or a fragment or variant thereof,comprise a polypeptide having the amino acid sequence of any one of theVH domains of the scFvs referred to in Table 2 and/or any one of the VLdomains of the scFvs referred to in Table 2. In preferred embodiments,antibodies of the present invention comprise the amino acid sequence ofa VH domain and VL domain from the same scFv selected from the groupconsisting of the scFvs referred to in Table 2 In alternativeembodiments, antibodies of the present invention comprise the amino acidsequence of a VH domain and a VL domain from different scFvs referred toin Table 2. Molecules comprising, or alternatively consisting of,antibody fragments or variants of the VH and/or VL domains of the scFvsreferred to in Table 2 that immunospecifically bind to a VEGF-2 are alsoencompassed by the invention, as are nucleic acid molecules encodingthese VH and VL domains, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of a VEGF-2,wherein said antibodies comprise, or alternatively consist of, apolypeptide having an amino acid sequence of any one, two, three, ormore of the VH CDRs contained in a VH domain of one or more scFvsreferred to in Table 2. In particular, the invention provides antibodiesthat immunospecifically bind a VEGF-2, comprising, or alternativelyconsisting of, a polypeptide having the amino acid sequence of a VH CDR1contained in a VH domain of one or more scFvs referred to in Table 2. Inanother embodiment, antibodies that immunospecifically bind a VEGF-2,comprise, or alternatively consist of, a polypeptide having the aminoacid sequence of a VH CDR2 contained in a VH domain of one or more scFvsreferred to in Table 2. In a preferred embodiment, antibodies thatimmunospecifically bind a VEGF-2, comprise, or alternatively consist ofa polypeptide having the amino acid sequence of a VH CDR3 contained in aVH domain of one or more scFvs referred to in Table 2. Moleculescomprising, or alternatively consisting of, these antibodies, orantibody fragments or variants thereof, that immunospecifically bind toVEGF-2 or a VEGF-2 fragment or variant thereof are also encompassed bythe invention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of a VEGF-2,wherein said antibodies comprise, or alternatively consist of, apolypeptide having an amino acid sequence of any one, two, three, ormore of the VL CDRs contained in a VL domain of one or more scFvsreferred to in Table 2. In particular, the invention provides antibodiesthat immunospecifically bind a VEGF-2, comprising, or alternativelyconsisting of, a polypeptide having the amino acid sequence of a VL CDR1contained in a VL domain of one or more scFvs referred to in Table 2. Inanother embodiment, antibodies that immunospecifically bind a VEGF-2,comprise, or alternatively consist of, a polypeptide having the aminoacid sequence of a VL CDR2 contained in a VL domain of one or more scFvsreferred to in Table 2. In a preferred embodiment, antibodies thatimmunospecifically bind a VEGF-2, comprise, or alternatively consist ofa polypeptide having the amino acid sequence of a VL CDR3 contained in aVL domain of one or more scFvs referred to in Table 2. Moleculescomprising, or alternatively consisting of, these antibodies, orantibody fragments or variants thereof, that immunospecifically bind toVEGF-2 or a VEGF-2 fragment or variant thereof are also encompassed bythe invention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that immunospecifically bind to a VEGF-2 polypeptide orpolypeptide fragment or variant of a VEGF-2, wherein said antibodiescomprise, or alternatively consist of, one, two, three, or more VH CDRsand one, two, three or more VL CDRs, as contained in a VH domain or VLdomain of one or more scFvs referred to in Table 2. In particular, theinvention provides for antibodies that immunospecifically bind to apolypeptide or polypeptide fragment or variant of a VEGF-2, wherein saidantibodies comprise, or alternatively consist of, a VH CDR1 and a VLCDR1, a VH CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and aVL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and aVH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or anycombination thereof, of the VH CDRs and VL CDRs contained in a VH domainor VL domain of one or more scFvs referred to in Table 2. In a preferredembodiment, one or more of these combinations are from the same scFv asdisclosed in Table 2. Molecules comprising, or alternatively consistingof, fragments or variants of these antibodies, that immunospecificallybind to VEGF-2 are also encompassed by the invention, as are nucleicacid molecules encoding these antibodies, molecules, fragments orvariants.

Nucleic Acid Molecules Encoding VEGF-2 Antibodies Corresponding toscFvs.

The present invention also provides for nucleic acid molecules,generally isolated, encoding an antibody of the invention (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof). In a specific embodiment, a nucleic acid moleculeof the invention encodes an antibody (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),comprising, or alternatively consisting of, a VH domain having an aminoacid sequence of any one of the VH domains of the scFvs referred to inTable 2 and a VL domain having an amino acid sequence of any one of theVL domains of the scFvs referred to in Table 2. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, a VHdomain having an amino acid sequence of any one of the VH domains of thescFvs referred to in Table 2 or a VL domain having an amino acidsequence of any one of the VL domains of the scFvs referred to in Table2.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the VH domains and/or VL domains) describedherein, which antibodies immunospecifically bind to a VEGF-2 orfragments or variant thereof. Standard techniques known to those ofskill in the art can be used to introduce mutations in the nucleotidesequence encoding a molecule of the invention, including, for example,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference VH domain, VHCDR1,VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a side chain with a similarcharge. Families of amino acid residues having side chains with similarcharges have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity (e.g., the ability to bind aVEGF-2).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind a VEGF-2) canbe determined using techniques described herein or by routinelymodifying techniques known in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that immunospecifically binds VEGF-2polypeptides or fragments or variants thereof, comprises, oralternatively consists of, an amino acid sequence encoded by anucleotide sequence that hybridizes to a nucleotide sequence that iscomplementary to that encoding one of the VH or VL domains of one ormore scFvs referred to in Table 2. under stringent conditions, e.g.,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., under highly stringent conditions, e.g.,hybridization to filter-bound nucleic acid in 6×SSC at about 45° C.followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., orunder other stringent hybridization conditions which are known to thoseof skill in the art (see, for example, Ausubel, F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3). Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a VEGF-2 polypeptide or fragments orvariants of a VEGF-2 polypeptide, comprises, or alternatively consistsof, a VH domain having an amino acid sequence that is at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical, to the amino acidsequence of a VH domain of an scFv referred to in Table 2.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a VEGF-2 polypeptide or fragments orvariants of a VEGF-2 polypeptide, comprises, or alternatively consistsof, a VL domain having an amino acid sequence that is at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical, to the amino acidsequence of a VL domain of an scFv referred to in Table 2.

Polynucleotides Encoding Antibodies

Antibodies of the invention (including antibody fragments or variants)can be produced by any method known in the art. For example, it will beappreciated that antibodies in accordance with the present invention canbe expressed in cell lines other than hybridoma cell lines. Sequencesencoding the cDNAs or genomic clones for the particular antibodies canbe used for transformation of a suitable mammalian or nonmammalian hostcells or to generate phage display libraries, for example. Additionally,polypeptide antibodies of the invention may be chemically synthesized orproduced through the use of recombinant expression systems.

One way to produce the antibodies of the invention would be to clone theVH and/or VL domains of the scFvs referred to in Table 2. In order toisolate the VH and VL domains from the hybridoma cell lines, PCR primersincluding VH or VL nucleotide sequences (See Example 32), may be used toamplify the expressed VH and VL sequences expressed by phage. The PCRproducts may then be cloned using vectors, for example, which have a PCRproduct cloning site consisting of a 5′ and 3′ single T nucleotideoverhang, that is complementary to the overhanging single adeninenucleotide added onto the 5′ and 3′ end of PCR products by many DNApolymerases used for PCR reactions. The VH and VL domains can then besequenced using conventional methods known in the art.

The cloned VH and VL genes may be placed into one or more suitableexpression vectors. By way of non-limiting example, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site may be used to amplifythe VH or VL sequences. Utilizing cloning techniques known to those ofskill in the art, the PCR amplified VH domains may be cloned intovectors expressing the appropriate immunoglobulin constant region, e.g.,the human IgG1 or IgG4 constant region for VH domains, and the humankappa or lambda constant regions for kappa and lambda VL domains,respectively.

Preferably, the vectors for expressing the VH or VL domains comprise apromoter suitable to direct expression of the heavy and light chains inthe chosen expression system, a secretion signal, a cloning site for theimmunoglobulin variable domain, immunoglobulin constant domains, and aselection marker such as neomycin. The VH and VL domains may also becloned into a single vector expressing the necessary constant regions.The heavy chain conversion vectors and light chain conversion vectorsare then co-transfected into cell lines to generate stable or transientcell lines that express full-length antibodies, e.g., IgG, usingtechniques known to those of skill in the art (See, for example, Guo etal., J. Clin. Endocrinol. Metab. 82:925-31 (1997), and Ames et al., J.Immunol. Methods 184:177-86 (1995) which are herein incorporated intheir entireties by reference).

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4 OR SEQ ID NO:18.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence(See Example 32) or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it may be useful to express the VH and VL domains ofone or more antibodies of the invention as single chain antibodies orFab fragments in a phage display library. For example, the cDNAsencoding the VH and VL domains of one or more antibodies of theinvention may be expressed in all possible combinations using a phagedisplay library, allowing for the selection of VH/VL combinations thatbind a VEGF-2 polypeptides with preferred binding characteristics suchas improved affinity or improved off rates. Additionally, VH and VLsegments—the CDR regions of the VH and VL domains of one or moreantibodies of the invention, in particular, may be mutated in vitro.Expression of VH and VL domains with “mutant” CDRs in a phage displaylibrary allows for the selection of VH/VL combinations that bind aVEGF-2 receptor polypeptides with preferred binding characteristics suchas improved affinity or improved off rates.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. The DNAencoding the VH and VL domains are joined together by an scFv linker byPCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13 and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to an antigen ofinterest (i.e., a VEGF-2 polypeptide or a fragment thereof) can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include, but are not limited to, those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18719; WO 93/11236; WO 95/15982; WO 95/20401; WO97/13844; and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,717; 5,780,225;5,658,727; 5,735,743 and 5,969,108; each of which is incorporated hereinby reference in its entirety.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis, by intracellular immunization (i.e., intrabody technology),or preferably, by recombinant expression techniques. Methods ofproducing antibodies include, but are not limited to, hybridomatechnology, EBV transformation, and other methods discussed herein aswell as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availability of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. Vectors that use glutamine synthase as the selectablemarker include, but are not limited to, the pEE6 expression vectordescribed in Stephens and Cockett, Nucl. Acids. Res 17:7110 (1989). Aglutamine synthase expression system and components thereof are detailedin PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are incorporated in their entireties by referenceherein. Additionally, glutamine synthase expression vectors that may beused according to the present invention are commercially available fromsuppliers, including, for example Lonza Biologics, Inc. (Portsmouth,N.H.). Expression and production of monoclonal antibodies using a GSexpression system in murine myeloma cells is described in Bebbington etal., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol.Prog. 11:1 (1995) which are incorporated in their entireties byreference herein.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

Antibody Conjugates

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994)Immnunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies, S. O. et al.(1992) PNAS 89:1428-1432; Fell, H. P. et al. (1991) J. Immunol.146:2446-2452 (said references incorporated by reference in theirentireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused to either the N-or C-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Antibodies of theinvention may also be fused to albumin (including but not limited torecombinant human serum albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is hereinincorporated by reference in its entirety. In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-x of human serum albumin, where x is an integer from 1 to 585and the albumin fragment has human serum albumin activity. In anotherpreferred embodiment, polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) are fused withpolypeptide fragments comprising, or alternatively consisting of, aminoacid residues 1-z of human serum albumin, where z is an integer from 369to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated byreference in its entirety. Polynucleotides encoding fusion proteins ofthe invention are also encompassed by the invention. Such fusionproteins may, for example, facilitate purification and may increasehalf-life in vivo. Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:18 may be fused or conjugated to the above antibody portions toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. Further, the polypeptidescorresponding to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO: 18 may be fusedor conjugated to the above antibody portions to facilitate purification.One reported example describes chimeric proteins consisting of the firsttwo domains of the human CD4-polypeptide and various domains of theconstant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide-linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (3H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸ Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴La, 175Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh,and ⁹⁷Ru.

In specific embodiments, VEGF-2 polypeptides of the invention areattached to macrocyclic chelators useful for conjugating radiometalions, including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm,to polypeptides. In a preferred embodiment, the radiometal ionassociated with the macrocyclic chelators attached to VEGF-2polypeptides of the invention is ¹¹¹In. In another preferred embodiment,the radiometal ion associated with the macrocyclic chelator attached toVEGF-2 polypeptides of the invention is ⁹⁰Y. In specific embodiments,the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to VEGF-2 polypeptideof the invention via a linker molecule. Examples of linker moleculesuseful for conjugating DOTA to a polypeptide are commonly known in theart—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90,1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmermanet al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporatedby reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361and 5,756,065, which disclose chelating agents that may be conjugated toantibodies, and methods for making and using them, are herebyincorporated by reference in their entireties. Though U.S. Pat. Nos.5,652,361 and 5,756,065 focus on conjugating chelating agents toantibodies, one skilled in the art could readily adapt the methodsdisclosed therein in order to conjugate chelating agents to otherpolypeptides.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The antibody conjugates of the invention can be used for modifying agiven biological response, the therapeutic agent or drug moiety is notto be construed as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as BIAcore analysis (see, e.g.,Example 33), FACS (Fluorescence activated cell sorter) analysis,immunofluorescence, immunocytochemistry, western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Exemplary immunoassays are described briefly below(but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I), orfragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of unlabeled antigen, and the detectionof the antibody bound to the labeled antigen. The affinity of theantibody of interest for a particular antigen and the binding off-ratescan be determined from the data by scatchard plot analysis. Competitionwith a second antibody can also be determined using radioimmunoassays.In this case, the antigen is incubated with antibody of interestconjugated to a labeled compound (e.g., compound labeled with ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody. This kind of competitive assay between two antibodies, mayalso be used to determine if two antibodies bind the same or differentepitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies (including antibody fragmentsor variants thereof) to a VEGF-2, or fragments of VEGF-2 BIAcore kineticanalysis comprises analyzing the binding and dissociation of antibodiesfrom chips with immobilized VEGF-2 on their surface as described indetail in Example 33.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, andanti-retroviral agents. In a highly preferred embodiment, antibodies ofthe invention may be administered alone or in combination with andanti-angiogenic agents. Generally, administration of products of aspecies origin or species reactivity (in the case of antibodies) that isthe same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M. More preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M or 10⁻⁸ M.Even more preferred binding affinities include those with a dissociationconstant or Kd less than 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Antibodies may further be used in an immunoassay to detect the presenceof tumors in certain individuals. Enzyme immunoassay can be performedfrom the blood sample of an individual. Elevated levels of VEGF-2 can beconsidered diagnostic of cancer.

Truncated versions of VEGF-2 can also be produced that are capable ofinteracting with wild type VEGF-2 to form dimers that fail to activateendothelial cell growth, therefore inactivating the endogenous VEGF-2.Or, mutant forms of VEGF-2 form dimers themselves and occupy the ligandbinding domain of the proper tyrosine kinase receptors on the targetcell surface, but fail to activate cell growth.

Alternatively, antagonists to the polypeptides of the present inventionmay be employed which bind to the receptors to which a polypeptide ofthe present invention normally binds. The antagonists may be closelyrelated proteins such that they recognize and bind to the receptor sitesof the natural protein, however, they are inactive forms of the naturalprotein and thereby prevent the action of VEGF-2 since receptor sitesare occupied. In these ways, the action of the VEGF-2 is prevented andthe antagonist/inhibitors may be used therapeutically as an anti-tumordrug by occupying the receptor sites of tumors which are recognized byVEGF-2 or by inactivating VEGF-2 itself. The antagonist/inhibitors mayalso be used to prevent inflammation due to the increased vascularpermeability action of VEGF-2. The antagonist/inhibitors may also beused to treat solid tumor growth, diabetic retinopathy, psoriasis andrheumatoid arthritis.

The antagonist/inhibitors may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinabove described.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples, certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 mg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 Fl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 mgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 EC are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.8:4057 (1980).

“Oligonucleotides” refer to either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands, which may bechemically synthesized. Such synthetic oligonucleotides have no 5′phosphate and thus will not ligate to another oligonucleotide withoutadding a phosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989), p. 146). Unless otherwiseprovided, ligation may be accomplished using known buffers andconditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 mg ofapproximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described bythe method of Graham, F. and Van der Eb, A., Virology 52:456-457 (1973).

EXAMPLES Example 1 Expression Pattern of VEGF-2 in Human Tissues andBreast Cancer Cell Lines

Northern blot analysis was carried out to examine the levels ofexpression of VEGF-2 in human tissues and breast cancer cell lines inhuman tissues. Total cellular RNA samples were isolated with RNAzol™ Bsystem (Biotecx Laboratories, Inc.). About 10 mg of total RNA isolatedfrom each breast tissue and cell line specified was separated on 1%agarose gel and blotted onto a nylon filter, (Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column from 5 Prime÷3Prime, Inc (Boulder, Colo.). The filter was then hybridized with aradioactive labeled full length VEGF-2 gene at 1,000,000 cpm/ml in 0.5 MNaPO₄ and 7% SDS overnight at 65° C. After washing twice at roomtemperature and twice at 60° C. with 0.5×SSC, 0.1% SDS, the filters werethen exposed at −70° C. overnight with an intensifying screen. A messageof 1.6 Kd was observed in 2 breast cancer cell lines. FIG. 5, lane #4represents a very tumorigenic cell line that is estrogen independent forgrowth.

Also, 10 mg of total RNA from 10 human adult tissues were separated onan agarose gel and blotted onto a nylon filter. The filter was thenhybridized with radioactively labeled VEGF-2 probe in 7% SDS, 0.5 MNaPO₄, pH 7.2; 1% BSA overnight at 65° C. Following washing in 0.2×SSCat 65° C., the filter was exposed to film for 24 days at −70° C. withintensifying screen. See FIG. 6.

Example 2 Expression of the Truncated Form of VEGF-2 (SEQ ID NO:4) by InVitro Transcription and Translation

The VEGF-2 cDNA was transcribed and translated in vitro to determine thesize of the translatable polypeptide encoded by the truncated form ofVEGF-2 and a partial VEGF-2 cDNA. The two inserts of VEGF-2 in thepBluescript SK vector were amplified by PCR with three pairs ofprimers, 1) M13-reverse and forward primers; 2) M13-reverse primer andVEGF primer F4; and 3) M13-reverse primer and VEGF primer F5. Thesequence of these primers are as follows.

M13-2 reverse primer: 5′-ATGCTTCCGGCTCGTATG-3′ (SEQ ID NO:9) Thissequence is located upstream of the 5′ end of the VEGF-2 cDNA insert inthe pBluescript vector and is in an anti-sense orientation as the cDNA.A T3 promoter sequence is located between this primer and the VEGF-2cDNA.

M13-2 forward primer: 5′GGGTTTTCCCAGTCACGAC-3′ (SEQ ID NO:10). Thissequence is located downstream of the 3′ end of the VEGF-2 cDNA insertin the pBluescript vector and is in an anti-sense orientation as thecDNA insert.

VEGF primer F4: 5′-CCACATGGTTCAGGAAAGACA-3′ (SEQ ID NO:11). Thissequence is located within the VEGF-2 cDNA in an anti-sense orientationfrom bp 1259-1239, which is about 169 bp away from the 3′ end of thestop codon and about 266 bp before the last nucleotide of the cDNA.

PCR reaction with all three pairs of primers produce amplified productswith T3 promoter sequence in front of the cDNA insert. The first andthird pairs of primers produce PCR products that encode the polypeptideof VEGF-2 shown in SEQ ID NO:4. The second pair of primers produce PCRproduct that misses 36 amino acids coding sequence at the C-terminus ofthe VEGF-2 polypeptide.

Approximately 0.5 mg of PCR product from first pair of primers, 1 mgfrom second pair of primers, 1 mg from third pair of primers were usedfor in vitro transcription/translation. The in vitrotranscription/translation reaction was performed in a 25 Fl of volume,using the T_(N)TJ Coupled Reticulocyte Lysate Systems (Promega, CAT#L4950). Specifically, the reaction contains 12.5 Fl of T_(N)T rabbitreticulocyte lysate 2 Fl of T_(N)T reaction buffer, 1 Fl of T3polymerase, 1 Fl of 1 mM amino acid mixture (minus methionine), 4 Fl of³⁵S-methionine (>1000 Ci/mmol, 10 mCi/ml), 1 Fl of 40 U/μl; RNasinribonuclease inhibitor, 0.5 or 1 mg of PCR products. Nuclease-free H₂Owas added to bring the volume to 25 Fl. The reaction was incubated at30° C. for 2 hours. Five microliters of the reaction product wasanalyzed on a 4-20% gradient SDS-PAGE gel. After fixing in 25%isopropanol and 10% acetic acid, the gel was dried and exposed to anX-ray film overnight at 70° C.

As shown in FIG. 7, PCR products containing the truncated VEGF-2 cDNA(i.e., as depicted in SEQ ID NO:3) and the cDNA missing 266 bp in the 3′un-translated region (3′-UTR) produced the same length of translatedproducts, whose molecular weights are estimated to be 38-40 dk (lanes 1and 3). The cDNA missing all the 3′UTR and missing sequence encoding theC-terminal 36 amino acids was translated into a polypeptide with anestimated molecular weight of 36-38 kd (lane 2).

Example 3 Cloning and Expression of VEGF-2 Using the BaculovirusExpression System

The DNA sequence encoding the VEGF-2 protein without 46 amino acids atthe N-terminus, see ATCC No. 97149, was amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

The 5′ primer has the sequence TGT AAT ACG ACT CAC TAT AGG GAT CCC GCCATG GAG GCC ACG GCT TAT GC (SEQ ID NO:12) and contains a BamnH1restriction enzyme site (in bold) and 17 nucleotide sequencecomplementary to the 5′ sequence of VEGF-2 (nt. 150-166).

The 3′ primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGG TCT (SEQID NO:13) and contains the cleavage site for the restriction enzyme XbaIand 18 nucleotides complementary to the 3′ sequence of VEGF-2, includingthe stop codon and 15 nt sequence before stop codon.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamnH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pAcGP67A baculovirus transfer vector (Pharmingen) at theBamH1 and XbaI sites. Through this ligation, VEGF-2 cDNA was cloned inframe with the signal sequence of baculovirus gp67 gene and was locatedat the 3′ end of the signal sequence in the vector. This is designatedpAcGP67A-VEGF-2.

To clone VEGF-2 with the signal sequence of gp67 gene to the pRG1 vectorfor expression, VEGF-2 with the signal sequence and some upstreamsequence were excised from the pAcGP67A-VEGF-2 plasmid at the Xhorestriction endonuclease site located upstream of the VEGF-2 cDNA and atthe XbaI restriction endonuclease site by XhoI and XbaI restrictionenzyme. This fragment was separated from the rest of vector on a 1%agarose gel and was purified using “Geneclean” kit. It was designatedF2.

The PRG1 vector (modification of pVL941 vector) is used for theexpression of the VEGF-2 protein using the baculovirus expression system(for review see: Summers, M. D. and Smith, G. E., “A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures,” TexasAgricultural Experimental Station Bulletin No. 1555, (1987)). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamH1, Sma1,XbaI, BglII and Asp718. A site for restriction endonuclease XhoI islocated upstream of BamH1 site. The sequence between XhoI and BamHI isthe same as that in PAcGp67A (static on tape) vector. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E. coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofcotransfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology 170:31-39 (1989).

The plasmid was digested with the restriction enzymes XboI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA was then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac gp67-VEGF-2) with the VEGF-2gene using the enzymes BamH1 and XbaI. The sequence of the clonedfragment was confirmed by DNA sequencing.

5 mg of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0 mg of acommercially available linearized baculovirus (“BaculoGoldJ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofectin method(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)).

1 mg of BaculoGoldJ virus DNA and 5 mg of the plasmid pBac gp67-VEGF-2were mixed in a sterile well of a microtiter plate containing 50 ml ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 ml Lipofectin plus 90 ml Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added dropwise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith, supra. As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the virus was added to the cells,blue stained plaques were picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses was then resuspended in anEppendorf tube containing 200 ml of Grace's medium. The agar was removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes were harvested andthen stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of 1. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 mCi of ³⁵S-methionine and 5 mCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Protein from the medium and cytoplasm of the Sf9 cells was analyzed bySDS-PAGE under non-reducing and reducing conditions. See FIGS. 8A and8B, respectively. The medium was dialyzed against 50 mM MES, pH 5.8.Precipitates were obtained after dialysis and resuspended in 100 mMNaCitrate, pH 5.0. The resuspended precipitate was analyzed again bySDS-PAGE and was stained with Coomassie Brilliant Blue. See FIG. 9.

The medium supernatant was also diluted 1:10 in 50 mM MES, pH 5.8 andapplied to an SP-650M column (1.0×6.6 cm, Toyopearl) at a flow rate of 1ml/min. Protein was eluted with step gradients at 200, 300 and 500 mMNaCl. The VEGF-2 was obtained using the elution at 500 mM. The eluatewas analyzed by SDS-PAGE in the presence or absence of reducing agent,b-mercaptoethanol and stained by Coomassie Brilliant Blue. See FIG. 10.

Example 4 Expression of Recombinant VEGF-2 in COS Cells

The expression of plasmid, VEGF-2-HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, an SV40 intron and polyadenylation site. A DNAfragment encoding the entire VEGF-2 precursor and a HA tag fused inframe to its 3′ end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (Wilson et al.,Cell 37:767 (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope. The plasmid construction strategy isdescribed as follows:

The DNA sequence encoding VEGF-2, ATCC No. 97149, was constructed by PCRusing two primers: the 5′ primer (CGC GGA TCC ATG ACT GTA CTC TAC CCA)(SEQ ID NO:14) contains a BamH1 site followed by 18 nucleotides ofVEGF-2 coding sequence starting from the initiation codon;

the 3′ sequence (CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTCGAG GCT CAT TTG TGG TCT 3′) (SEQ ID NO:15) contains complementarysequences to an XbaI site, HA tag, XhoI site, and the last 15nucleotides of the VEGF-2 coding sequence (not including the stopcodon).

Therefore, the PCR product contains a BamHI site, coding sequencefollowed by an XhoI restriction endonuclease site and HA tag fused inframe, a translation termination stop codon next to the HA tag, and anXbaI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp,were digested with BamH1 and XbaI restriction enzyme and ligated. Theligation mixture was transformed into E. coli strain SURE (StratageneCloning Systems, La Jolla, Calif. 92037) the transformed culture wasplated on ampicillin media plates and resistant colonies were selected.Plasmid DNA was isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment. For expression of therecombinant VEGF-2, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the VEGF-2-HA protein was detected byradiolabelling and immunoprecipitation method (E. Harlow and D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media was then collected and cells were lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson et al., Cell 37:767 (1984)). Both celllysate and culture media were precipitated with an HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

Example 5 The Effect of Partially Purified VEGF-2 Protein on the Growthof Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) were seeded at2−5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumwas replaced with M199 containing 10% FBS, 8 units/ml heparin. VEGF-2protein of SEQ ID NO. 2 minus the initial 45 amino acid residues, (VEGF)and basic FGF (bFGF) were added, at the concentration shown. On days 4and 6, the medium was replaced. On day 8, cell number was determinedwith a Coulter Counter (See FIG. 12).

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 induced proliferation of HUVECcells.

Example 6 The Effect of Purified VEGF-2 Protein on the Growth ofVascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) were seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, 50 units/ml endothelial cellgrowth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium wasreplaced with M199 containing 10% FBS, 8 units/ml heparin. PurifiedVEGF-2 protein of SEQ ID NO:2 minus initial 45 amino acid residues wasadded to the medium at this point. On days 4 and 6, the medium wasreplaced with fresh medium and supplements. On day 8, cell number wasdetermined with a Coulter Counter (See FIG. 13).

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 induced proliferation of HUVECcells.

Example 7 Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA 7:219-225 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 8 Expression of VEGF-2 mRNA in Human Fetal and Adult Tissues

Experimental Design

Northern blot analysis was carried out to examine the levels ofexpression of VEGF-2 mRNA in human fetal and adult tissues. A cDNA probecontaining the entire nucleotide sequence of the VEGF-2 protein waslabeled with ³²P using the rediprime° DNA labeling system (Amersham LifeScience), according to the manufacturer's instructions. After labeling,the probe was purified using a CHROMA SPIN-100* column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe was then used to examine varioushuman tissues for VEGF-2 mRNA.

A Multiple Tissue Northern (MTN) blot containing various human tissues(Fetal Kidney, Fetal Lung, Fetal Liver, Brain, Kidney, Lung, Liver,Spleen, Thymus, Bone Marrow, Testes, Placenta, and Skeletal Muscle) wasobtained from Clontech. The MTN blot was examined with the labeled probeusing ExpressHyb* hybridization solution (Clontech) according tomanufacturer's protocol number PT1190-1. Following hybridization andwashing, the blot was exposed to film at −70° C. overnight with anintensifying screen and developed according to standard procedures.

Results

Expression of VEGF-2 mRNA is abundant in vascular smooth muscle andseveral highly vascularized tissues. VEGF-2 is expressed atsignificantly higher levels in tissues associated with hematopoetic orangiogenic activities, i.e. fetal kidney, fetal lung, bone marrow,placental, spleen and lung tissue. The expression level of VEGF-2 is lowin adult kidney, fetal liver, adult liver, testes; and is almostundetectable in fetal brain, and adult brain (See FIGS. 14A-B).

In primary cultured cells, the expression of VEGF-2 mRNA is abundant invascular smooth muscle cells and dermal fibroblast cells, but much lowerin human umbilical vein endothelial cells (see FIG. 15). This mRNAdistribution pattern is very similar to that of VEGF.

Example 9 Construction of Amino Terminal and Carboxy Terminal DeletionMutants

In order to identify and analyze biologically active VEGF-2polypeptides, a panel of deletion mutants of VEGF-2 was constructedusing the expression vector pHE4a.

1. Construction of VEGF-2 T103-L215 in pHE4

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG. 1 or SEQID NO: 18) into the E. coli protein expression vector, pHE4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (Nde I/START and 18 nt of coding sequence): (SEQ ID NO: 19)5′-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3′; 3′Primer (Asp718, STOP, and 15 nt of coding sequence): (SEQ ID NO: 20)5′-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3′.

The above described 5′ primer (SEQ ID NO: 19), incorporates an NdeIrestriction site and the above described 3′ Primer (SEQ ID NO:20),incorporates an Asp718 restriction site. The 5′ primer (SEQ ID NO:19)also contains an ATG sequence adjacent and in frame with the VEGF-2coding region to allow translation of the cloned fragment in E. coli,while the 3′ primer (SEQ ID NO:20) contains one stop codon(preferentially utilized in E. coli) adjacent and in frame with theVEGF-2 coding region which ensures correct translational termination inE. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419 in SEQ ID NO:18) as, for example,constructed in Example 3 as template. The resulting amplicon wasrestriction digested with NdeI and Asp718 and subcloned into NdeI/Asp718digested pHE4a expression vector.

2. Construction of VEGF-2 T103-R227 in pHE4

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-R227 (amino acids 103 to 227 in FIG. 1 or SEQID NO:18) into the E. coli protein expression vector, pHE4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (Nde I/START and 18 nt of coding sequence): (SEQ ID NO: 19)5′-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3′; 3′Primer (Asp 718, STOP, and 15 nt of coding sequence): (SEQ ID NO: 21)5′-GCA GCA GGT ACC TCA ACG TCT AAT AAT GGA-3′,

In the case of the above described primers, an NdeI or Asp718restriction site was incorporated the 5′ primer and 3′ primer,respectively. The 5′ primer (SEQ ID NO:19) also contains an ATG sequenceadjacent and in frame with the VEGF-2 coding region to allow translationof the cloned fragment in E. coli, while the 3′ Primer (SEQ ID NO:21)contains one stop codon (preferentially utilized in E. coli) adjacentand in frame with the VEGF-2 coding region which ensures correcttranslational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419 in SEQ ID NO:18) as, for example,constructed in Example 3, as template. The resulting amplicon wasrestriction digested with NdeI and Asp718 and subcloned into NdeI/Asp718digested pHE4a protein expression vector.

3. Construction of VEGF-2 T103-L215 in pA2GP

In this illustrative example, the plasmid shuttle vector pA2 GP is usedto insert the cloned DNA encoding the N-terminal and C-terminal deletedVEGF-2 protein (amino acids 103-215 in FIG. 1 or SEQ ID NO:18), into abaculovirus to express the N-terminal and C-terminal deleted VEGF-2protein, using a baculovirus leader and standard methods as described inSummers et al., A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by the secretory signal peptide (leader) of thebaculovirus gp67 protein and convenient restriction sites such as BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is used for efficient polyadenylation. For easy selection ofrecombinant virus, the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that expresses the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM4, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the VEGF-2 protein without 102 amino acids atthe N-terminus and without 204 amino acids at the C-terminus in FIG. 1,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene.

The 5′ primer has the sequence 5′-GCA GCA GGA TCC CAC AGA AGA GAC TATAAA-3′ (SEQ ID NO:22) containing the BamHI restriction enzyme site (inbold) followed by 1 spacer nt to stay in-frame with the vector-suppliedsignal peptide, and 17 nt of coding sequence bases of VEGF-2 protein.The 3′ primer has the sequence, 5′-GCA GCA TCT AGA TCA CAG TTT AGA CATGCA-3′ (SEQ ID NO:23) containing the XbaI restriction site (in bold)followed by a stop codon and 17 nucleotides complementary to the 3′coding sequence of VEGF-2.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pA2 GP baculovirus transfer vector (Supplier) at the BamH1and XbaI sites. Through this ligation, VEGF-2 cDNA representing theN-terminal and C-terminal deleted VEGF-2 protein (amino acids 103-215 inFIG. 1 or SEQ ID NO:18) was cloned in frame with the signal sequence ofbaculovirus GP gene and was located at the 3′ end of the signal sequencein the vector. This is designated pA2GPVEGF-2.T103-L215.

4. Construction of VEGF-2 T103-R227 in pA2GP

The cDNA sequence encoding the VEGF-2 protein without 102 amino acids atthe N-terminus and without 192 amino acids at the C-terminus in FIG. 1(i.e., amino acids 103-227 of SEQ ID NO:18) was amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene.

The 5′-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC TGC-3′ primer hasthe sequence (SEQ ID NO:24) containing the BamHI restriction enzyme site(in bold) followed by 1 spacer nt to stay in-frame with thevector-supplied signal peptide, and 26 nt of coding sequence bases ofVEGF-2 protein. The 3′ primer has the sequence 5′-GCA GCA TCT AGA TCAACG TCT AAT AAT GGA ATG AAC-3′ (SEQ ID NO:25) containing the XbaIrestriction site (in bold) followed by a stop codon and 21 nucleotidescomplementary to the 3′ coding sequence of VEGF-2.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pA2 GP baculovirus transfer vector (Supplier) at the BamH1and XbaI sites. Through this ligation, VEGF-2 cDNA representing theN-terminal and C-terminal deleted VEGF-2 protein (amino acids 103-227 inFIG. 1 or SEQ ID NO: 18) was cloned in frame with the signal sequence ofbaculovirus GP gene and was located at the 3′ end of the signal sequencein the vector. This construct is designated pA2GPVEGF-2.T103-R227.

5. Construction of VEGF-2 in pC1

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3Nintron, the polyadenylation and termination signal of the ratpreproinsulin gene.

The vector pC1 is used for the expression of VEGF-2 protein. Plasmid pC1is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146].Both plasmids contain the mouse DHFR gene under control of the SV40early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-4470)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Pvull,and Nrul. Behind these cloning sites the plasmid contains translationalstop codons in all three reading frames followed by the 3N intron andthe polyadenylation site of the rat preproinsulin gene. Other highefficient promoters can also be used for the expression, e.g., the humanb-actin promoter, the SV40 early or late promoters or the long terminalrepeats from other retroviruses, e.g., HIV and HTLVI. For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well.

Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding VEGF-2, ATCC Accession No. 97149, wasconstructed by PCR using two primers corresponding to the 5′ and 3′ endsof the VEGF-2 gene:

the 5′ Primer (5′-GAT CGA TCC ATC ATG CAC TCG CTG GGC TTC TTC TCT GTGGCG TGT TCT CTG CTC G-3′ (SEQ ID NO:26)) contains a Klenow-filled BamHIsite and 40 nt of VEGF-2 coding sequence starting from the initiationcodon;

the 3′ primer (5′-GCA GGG TAC GGA TCC TAG ATT AGC TCA TTT GTG GTC TTT-3′(SEQ ID NO:27)) contains a BamHI site and 16 nt of VEGF-2 codingsequence not including the stop codon.

The PCR amplified DNA fragment is isolated from a 1% agarose gel asdescribed above and then digested with the endonuclease BamHI and thenpurified again on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 cells are then transformed and bacteria identified that containedthe plasmid pC1. The sequence and orientation of the inserted gene isconfirmed by DNA sequencing. This construct is designated pC1VEGF-2.

6. Construction of pC4SigVEGF-2 T103-L215

Plasmid pC4Sig is plasmid pC4 (Accession No. 209646) containing a humanIgG Fc portion as well as a protein signal sequence.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 215 in FIG. 1 or SEQID NO:18) into pC4 μg, two oligonucleotide primers complementary to thedesired region of VEGF-2 were synthesized with the following basesequence:

5′ Primer (Bam HI and 26 nt of coding sequence): (SEQ ID NO: 34)5′-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3′; 3′Primer (Xba I, STOP, and 15 nt of coding sequence): (SEQ ID NO: 35)5′-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG GCA G-3′.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as, for example, constructed in Example 3,as template. The resulting amplicon was restriction digested with BamHIand XbaI and subcloned into BamHI/XbaI digested pC4 μg vector.

7. Construction of pC4SigVEGF-2 T103-R227

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 T103-L215 (amino acids 103 to 227 in FIG. 1 or SEQID NO:18) into pC4Sig, two oligonucleotide primers complementary to thedesired region of VEGF-2 were synthesized with the following basesequence:

5′ Primer (Bam HI and 26 nt of coding sequence): (SEQ ID NO: 34)5′-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3′; 3′Primer (Xba I, STOP, and 21 nt of coding sequence): (SEQ ID NO: 25)5′-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3′.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as, for example, constructed in Example 3,as template. The resulting amplicon was restriction digested with BamHIand XbaI and subcloned into BamHI/XbaI digested pC4Sig vector.

8. Construction of pC4VEGF-2 M1-M263

The expression vector pC4 contains the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vector contains in addition the 3N intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 M1-M263 protein (amino acids 1-263 in FIG. 1 or SEQ IDNO:18) is inserted into the plasmid vector pC4 to express the C-terminaldeleted VEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-M263 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 28)5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3′; 3′ Primer(SEQ ID NO: 29) 5′-GAC TGG TAC CTT ATC ACA TAA AAT CTT CCT GAG CC-3′.

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamH1and Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector. This construct is designated pC4VEGF-2 M1-M263.

9. Construction of pC4VEGF-2 M1-D311

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 M1-D311 protein (amino acids 1-311 in FIG. 1 or SEQ IDNO:18) is inserted into the plasmid vector pC4 to express the C-terminaldeleted VEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-D311 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 30)5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3′; 3′ Primer(SEQ ID NO: 31) 5′-GAC TGG TAC CTT ATC AGT CTA GTT CTT TGT GGG G-3′.

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamHIand Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector.

10. Construction of pC4VEGF-2 M1-Q367

In this illustrative example, the cloned DNA encoding the C-terminaldeleted VEGF-2 M1-Q367 protein (amino acids 1-367 in SEQ ID NO:18) isinserted into the plasmid vector pC4 to express the C-terminal deletedVEGF-2 protein.

To permit Polymerase Chain Reaction directed amplification andsub-cloning of VEGF-2 M1-Q367 into the expression vector, pC4, twooligonucleotide primers complementary to the desired region of VEGF-2were synthesized with the following base sequence:

5′ Primer (SEQ ID NO: 32)5′-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3′; 3′ Primer(SEQ ID NO: 33) 5′-GAC TGG TAC CTC ATT ACT GTG GAC TTT CTG TAC ATT C-3′.

In the case of the above described 5′ primer, an BamH1 restriction sitewas incorporated, while in the case of the 3′ primer, an Asp718restriction site was incorporated. The 5′ primer also contains 6 nt, 20nt of VEGF-2 coding sequence, and an ATG sequence adjacent and in framewith the VEGF-2 coding region to allow translation of the clonedfragment in E. coli, while the 3′ primer contains 2 nt, 20 nt of VEGF-2coding sequence, and one stop codon (preferentially utilized in E. coli)adjacent and in frame with the VEGF-2 coding region which ensurescorrect translational termination in E. coli.

The Polymerase Chain Reaction was performed using standard conditionswell known to those skilled in the art and the nucleotide sequence forthe mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3as template. The resulting amplicon was restriction digested with BamH1and Asp718 and subcloned into BamH1/Asp718 digested pC4 proteinexpression vector. This construct is designated pC4VEGF-2 M1-Q367.

Example 10 Transient Expression of VEGF-2 Protein in COS-7 Cells

Experimental Design

Expression of the VEGF-2-HA fusion protein from the construct made inExample 4, for example, was detected by radiolabeling andimmunoprecipitation, using methods described in, for example Harlow andcolleagues (Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988)). To this end, twodays after transfection, the cells were labeled by incubation in mediacontaining 35S-cysteine for 8 hours. The cells and the media werecollected, and the cells were washed and then lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson andcolleagues (supra). Proteins were precipitated from the cell lysate andfrom the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then were analyzed by SDS-PAGE andautoradiography.

Results

As shown in FIGS. 16A-B, cells transfected with pcDNA1 VEGF-2HA secreteda 56 kd and a 30 kd protein. The 56 kd protein, but not the 30 kdprotein, could also be detected in the cell lysate but is not detectedin controls. This suggests the 30 kd protein is likely to result fromcleavage of the 56 kd protein. Since the HA-tag is on the C-terminus ofVEGF-2, the 30 kd protein must represent the C-terminal portion of thecleaved protein, whereas the N-terminal portion of the cleaved proteinwould not be detected by immunoprecipitation. These data indicate thatVEGF-2 protein expressed in mammalian cells is secreted and processed.

Example 11 Stimulatory Effect of VEGF-2 on Proliferation of VascularEndothelial Cells

Experimental Design

Expression of VEGF-2 is abundant in highly vascularized tissues.Therefore the role of VEGF-2 in regulating proliferation of severaltypes of endothelial cells was examined.

Endothelial Cell Proliferation Assay

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)₂H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells were seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and allowedto attach overnight. After serum-starvation for 12 hours in 0.5% FBS,conditions (bFGF, VEGF₁₆₅ or VEGF-2 in 0.5% FBS) with or without Heparin(8 U/ml) were added to wells for 48 hours. 20 mg of MTS/PMS mixture(1:0.05) were added per well and allowed to incubate for 1 hour at 37°C. before measuring the absorbance at 490 nm in an ELISA plate reader.Background absorbance from control wells (some media, no cells) wassubtracted, and seven wells were performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994)

Results

VEGF-2 stimulated proliferation of human umbilical vein endothelialcells (HUVEC) and dermal microvascular endothelial cells slightly (FIGS.17 and 18). The stimulatory effect of VEGF-2 is more pronounced onproliferation of endometrial and microvascular endothelial cells (FIG.19). Endometrial endothelial cells (HEEC) demonstrated the greatestresponse to VEGF-2 (96% of the effect of VEGF on microvascularendothelial cells). The response of microvascular endothelial cells(HMEC) to VEGF-2 was 73% compared to VEGF. The response of HUVEC andBAEC (bovine aortic endothelial cells) to VEGF-2 was substantially lowerat 10% and 7%, respectively. The activity of VEGF-2 protein has variedbetween different purification runs with the stimulatory effect ofcertain batches on HUVEC proliferation being significantly higher thanthat of other batches.

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 induced proliferation of endothelialcells.

Example 12 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation

VEGF-2 expression is high in vascular smooth muscle cells. Smooth muscleis an important therapeutic target for vascular diseases, such asrestenosis. To evaluate the potential effects of VEGF-2 on smooth musclecells, the effect of VEGF-2 on human aortic smooth muscle cell (HAoSMC)proliferation was examined.

Experimental Design

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4° C. for 2 h after exposing to denaturingsolution and then with the streptavidin-peroxidase and diaminobenzidine.After counterstaining with hematoxylin, the cells are mounted formicroscopic examination, and the BrdUrd-positive cells are counted. TheBrdUrd index is calculated as a percent of the BrdUrd-positive cells tothe total cell number. In addition, the simultaneous detection of theBrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performedfor individual cells by the concomitant use of bright field illuminationand dark field-UV fluorescent illumination. See, Hayashida et al., J.Biol. Chem. 6; 271(36):21985-21992 (1996).

Results

VEGF-2 has an inhibitory effect on proliferation of vascular smoothmuscle cells induced by PDGF, but not by Fetal Bovine Serum (FBS) (FIG.20).

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 inhibition of vascular smooth musclecell proliferation.

Example 13 Stimulation of Endothelial Cell Migration

Endothelial cell migration is an important step involved inangiogenesis.

Experimental Design

This example will be used to explore the possibility that VEGF-2 maystimulate lymphatic endothelial cell migration. Currently, there are nopublished reports of such a model. However, we will be adapting a modelof vascular endothelial cell migration for use with lymphaticendothelial cells essentially as follows:

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W.,Goodwin, R. H. J., and Leonard, E. J. “A 48 well micro chemotaxisassembly for rapid and accurate measurement of leukocyte migration.” J.Immunological Methods 1980; 33:239-247). Polyvinylpyrrolidone-freepolycarbonate filters with a pore size of 8 um (Nucleopore Corp.Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours atroom temperature and dried under sterile air. Test substances arediluted to appropriate concentrations in M199 supplemented with 0.25%bovine serum albumin (BSA), and 25 ul of the final dilution is placed inthe lower chamber of the modified Boyden apparatus. Subconfluent, earlypassage (2-6) HUVEC or BMEC cultures are washed and trypsinized for theminimum time required to achieve cell detachment. After placing thefilter between lower and upper chamber, 2.5×10⁵ cells suspended in 50 ulM199 containing 1% FBS are seeded in the upper compartment. Theapparatus is then incubated for 5 hours at 37° C. in a humidifiedchamber with 5% CO₂ to allow cell migration. After the incubationperiod, the filter is removed and the upper side of the filter with thenon-migrated cells is scraped with a rubber policeman. The filters arefixed with methanol and stained with a Giemsa solution (Diff-Quick,Baxter, McGraw Park, Ill.). Migration is quantified by counting cells ofthree random high-power fields (40×) in each well, and all groups areperformed in quadruplicate.

Results

In an assay examining HUVEC migration using a 43-well microchemotaxischamber, VEGF-2 was able to stimulate migration of HUVEC (FIGS. 21A-B).

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 induced migration of endothelialcells.

Example 14 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. VEGF-1 has beendemonstrated to induce nitric oxide production by endothelial cells inresponse to VEGF-1. As a result, VEGF-2 activity can be assayed bydetermining nitric oxide production by endothelial cells in response toVEGF-2.

Experimental Design

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of VEGF-1 and VEGF-2. Nitric oxide in themedium is determined by use of the Griess reagent to measure totalnitrite after reduction of nitric oxide-derived nitrate by nitratereductase. The effect of VEGF-2 on nitric oxide release was examined onHUVEC.

Briefly, NO release from cultured HUVEC monolayer was measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementswas performed according to the following equation: 2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2 K₂SO₄

The standard calibration curve was obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO was previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium was removed and HUVECswere washed twice with Dulbecco's phosphate buffered saline. The cellswere then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates were kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe was inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) was used as apositive control. The amount of released NO was expressed as picomolesper 1×10⁶ endothelial cells. All values reported were means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

Results

VEGF-2 was capable of stimulating nitric oxide release on HUVEC (FIG.22) to a higher level than VEGF. This suggested that VEGF-2 may modifyvascular permeability and vessel dilation.

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 induced nitric oxide release fromendothelial cells.

Example 15 Effect of VEGF-2 on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

Experimental Design

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications'CADMEC Growth Medium and used at passage 5. For thein vitro angiogenesis assay, the wells of a 48-well cell culture plateare coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Cord FormationMedium containing control buffer or the protein of the invention (0.1 to100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like cords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-estradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

Results

It has been observed that VEGF-2 inhibits cord formation similar to IFNawhich also stimulates endothelial cell proliferation (FIG. 23). Thisinhibitory effect may be a secondary effect of endothelial proliferationwhich is mutually exclusive with the cord formation process.

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 inhibition of cord formation.

Example 16 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of VEGF-2 to stimulate angiogenesis in CAMwas examined.

Experimental Design

Embryos

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) were incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos was studied with the following methods.

CAM Assay

On Day 4 of development, a window was made into the egg shell of chickeggs. The embryos were checked for normal development and the eggssealed with cellotape. They were further incubated until Day 13.Thermanox coverslips (Nunc, Naperville, Ill.) were cut into disks ofabout 5 mm in diameter. Sterile and salt-free growth factors weredissolved in distilled water and about 3.3 mg/5 ml was pipetted on thedisks. After air-drying, the inverted disks were applied on CAM. After 3days, the specimens were fixed in 3% glutaraldehyde and 2% formaldehydeand rinsed in 0.12 M sodium cacodylate buffer. They were photographedwith a stereo microscope [Wild M8] and embedded for semi- and ultrathinsectioning as described above. Controls were performed with carrierdisks alone.

Results

This data demonstrates that VEGF-2 can stimulate angiogenesis in the CAMassay nine-fold compared to the untreated control. However, thisstimulation is only 45% of the level of VEGF stimulation (FIG. 24).

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 stimulation of angiogenesis in theCAM assay.

Example 17 Angiogenesis Assay Using a Matrigel Implant in Mouse

Experimental Design

In order to establish an in vivo model for angiogenesis to test proteinactivities, mice and rats have been implanted subcutaneously withmethylcellulose disks containing either 20 mg of BSA (negative control)and 1 mg of bFGF and 0.5 mg of VEGF-1 (positive control).

It appeared as though the BSA disks contained little vascularization,while the positive control disks showed signs of vessel formation. Atday 9, one mouse showed a clear response to the bFGF.

Results

Both VEGF proteins appeared to enhance Matrigel cellularity by a factorof approximately 2 by visual estimation.

An additional 30 mice were implanted with disks containing BSA, bFGF,and varying amounts of VEGF-1, VEGF-2-B8, and VEGF-2-C4. Each mousereceived two identical disks, rather than one control and oneexperimental disk.

Samples of all the disks recovered were immunostained with VonWillebrand's factor to detect for the presence of endothelial cells inthe disks, and flk-1 and flt-4 to distinguish between vascular andlymphatic endothelial cells. However, definitive histochemical analysisof neovascularization and lymphangiogenesis could not be determined.

Additionally, one of skill in the art could readily modify the aboveprotocol to test the effect of agonists and/or antagonists of VEGF-2(e.g., VEGF-2 antibodies) on VEGF-2 modulated angiogenesis.

Example 18 Rescue of Ischemia in Rabbit Lower Limb Model

Experimental Design

To study the in vivo effects of VEGF-2 on ischemia, a rabbit hindlimbischemia model was created by surgical removal of one femoral artery asdescribed previously (Takeshita, S. et al., Am J Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al. Am J Pathol 147:1649-1660 (1995)). An interval of10 days was allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb was transfected with 500 mg naked VEGF-2expression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al. HumGene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin. Invest. 90:936-944 (1992)). When VEGF-2 was used in the treatment, a single bolusof 500 mg VEGF-2 protein or control was delivered into the internaliliac artery of the ischemic limb over a period of 1 min. through aninfusion catheter. On day 30, various parameters were measured in theserabbits.

Results

Both VEGF-2 protein (FIG. 25A) and naked expression plasmid (FIG. 25B)were able to restore the following parameters in the ischemic limb.Restoration of blood flow, angiographic score seem to be slightly moreby administration of 500 mg plasmid compared with by 500 mg protein(FIG. 25C) The extent of the restoration is comparable with that by VEGFin separate experiments (data not shown). A vessel dilator was not ableto achieve the same effect, suggesting that the blood flow restorationis not simply due to a vascular dilation effect.

1. BP Ratio (FIGS. 25A-C)

The blood pressure ratio of systolic pressure of the ischemic limb tothat of normal limb.

2. Blood Flow and Flow Reserve (FIGS. 25D-I)

Resting FL: the blood flow during un-dilated condition

Max FL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount)

Flow Reserve is reflected by the ratio of max FL:resting FL.

3. Angiographic Score (FIGS. 25J-L)

This is measured by the angiogram of collateral vessels. A score wasdetermined by the percentage of circles in an overlaying grid that withcrossing opacified arteries divided by the total number m the rabbitthigh.

4. Capillary Density (FIGS. 25M-O)

The number of collateral capillaries determined in light microscopicsections taken from hindlimbs.

As discussed, VEGF-2 is processed to an N-terminal and a C-terminalfragment which are co-purified. The N-terminal fragment contains theintact putative functional domain and may be responsible for thebiologic activity.

Example 19 Effect of VEGF-2 on Vasodilation

As described above, VEGF-2 can stimulate NO release, a mediator ofvascular endothelium dilation. Since dilation of vascular endothelium isimportant in reducing blood pressure, the ability of VEGF-2 to affectthe blood pressure in spontaneously hypertensive rats (SHR) wasexamined. VEGF-2 caused a dose-dependent decrease in diastolic bloodpressure (FIGS. 26 a and b). There was a steady decline in diastolicblood pressure with increasing doses of VEGF-2 which attainedstatistical significance when a dose of 300 mg/kg was administered. Thechanges observed at this dose were not different than those seen withacetylcholine (0.5 mg/kg). Decreased mean arterial pressure (MAP) wasobserved as well (FIGS. 26 c and d). VEGF-2 (300 mg/kg) andacetylcholine reduced the MAP of these SHR animals to normal levels.

Additionally, increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) ofthe B8, C5, and C4 preps of VEGF-2 were administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis was performed with a paired t-test andstatistical significance was defined as p<0.05 vs. the response tobuffer alone.

Studies with VEGF-2 (C5 prep) revealed that although it significantlydecreased the blood pressure, the magnitude of the response was not asgreat as that seen with VEGF-2 (B8 prep) even when used at a dose of 900mg/kg.

Studies with VEGF-2 (C4 preparation) revealed that this CHO expressedprotein preparation yielded similar results to that seen with C5 (i.e.statistically significant but of far less magnitude than seen with theB8 preparation) (see FIGS. 26A-D).

As a control and since the C4 and C5 batches of VEGF-2 yielded minor,but statistically significant, changes in blood pressure, experimentswere performed experiments with another CHO-expressed protein, M-CIF.Administration of M-CIF at doses ranging from 10-900 mg/kg produced nosignificant changes in diastolic blood pressure. A minor statisticallysignificant reduction in mean arterial blood pressure was observed atdoses of 100 and 900 mg/kg but no dose response was noted. These resultssuggest that the reductions in blood pressure observed with the C4 andC5 batches of VEGF-2 were specific, i.e. VEGF-2 related.

Example 20 Rat Ischemic Skin Flap Model

Experimental Design

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. VEGF-2 expression, during the skin ischemia, is studied usingin situ hybridization.

The study in this model is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

c) Topical treatment with VEGF-2 of the excisional wounds (day 0, 1, 2,3, 4 post-wounding) at the following various dosage ranges: 1 mg to 100mg.

d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

Example 21 Peripheral Arterial Disease Model

Angiogenic therapy using VEGF-2 has been developed as a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases.

Experimental Design

The experimental protocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) VEGF-2 protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of VEGF-2 expression andhistology. Biopsy is also performed on the other side of normal muscleof the contralateral hindlimb.

Example 22 Ischemic Myocardial Disease Model

VEGF-2 is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of VEGF-2 expression isinvestigated in situ.

Experimental Design

The experimental protocol includes:

a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) VEGF-2 protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-4 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

Example 23 Rat Corneal Wound Healing Model

This animal model shows the effect of VEGF-2 on neovascularization.

Experimental Design

The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

b) Inserting a spatula below the lip of the incision facing the outercorner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 mg-500 mg VEGF-2, within thepocket.

e) VEGF-2 treatment can also be applied topically to the corneal woundsin a dosage range of 20 mg-500 mg (daily treatment for five days).

Alternative Protocol

In this protocol, the VEGF-2 polypeptide and/or VEGF-2 antibodies aredelivered to the rat cornea using a filter disk which is inserted intothe cornea as described below.

Filter Disk Preparation.

Sterile cornea filter disks are stamped from 0.45 μm pore size MilliporeHAWP01300 filters using a sterile 20 G needle with the standard bevelcut off and a machined bevel ground around the flattened tip underbiosafety hood. Stamped disks are removed from the 20 G needle by meansof a 24 G stylet. VEGF-2 polypeptides and or VEGF-2 antibody solutionare prepared in sterile filtered 1×TBS (50 mM Tris-HCl pH 7.4/150 mMNaCl) as described below. The control groups will receive 1×TBS or flagpeptide only.

Cornea Surgery and Insertion of Filter Disk.

Generally, 20 Sprague Dawley rats weighing 175-200 grams is used forthese experiments. On the day of surgery, each animal is anesthetizedwith ketamine (50 mg/kg im; Phoenix # NDC 57319-291-02), xylazine (10mg/kg im; Phoenix # NDC 57319-326-26), and acepromazine (1.0 mg/kg im;Fermenta #117-531). After the rat is anesthetized, the vibrissae istrimmed, and the rat is injected with 0.5 mg/kg atropine sulfate (RBI#A-105; Lot #69H0545). The rat is wrapped in sterile surgical drape.Sterile gloves is used for the surgical procedure. The surgical field(eye plus surrounding fur) is rinsed with saline, followed by 5%povidone-iodine (Perdue Frederick #H8151-K97; Lot #6H31). The eye isthen be rinsed with sterile saline and 2 drops of 2% lidocaine HCl(Phoenix #NDC-57319-093-05; Lot #0080991) are dropped onto the eye. Eyesare irrigated with saline throughout the procedure to preventdesiccation. An incision is made with a sterile #15 scalpel blade 2 mmfrom the corneal limbus. The incision is made approximately half waythrough the thickness of the cornea. After the incision is made, sterilemicrosurgical scissors are used to create a pocket that extends from thepoint of the incision to approximately 0.75 mm from the limbus. Thepresoaked disk (soaked in a sterile petri dish on ice overnight in 20 μLof the respective test solution) is inserted into this pocket so thatthe leading edge of the disk is 1 mm from the limbus.

After the surgery is complete, the eyelid is closed, and gently heldtogether with a microaneurysm clamp. The rat is then be turned over, andthe procedure starting at step #6 is repeated. After both eyes arefinished, the rat is placed in an isolation cage to wake up. As soon asthe rat begins to regain consciousness, the microaneurysm clamps areremoved.

Imaging:

Five days following the surgery, the rat is dosed with 0.5 mg/kgatropine. Upon observation of mydriasis, the animal is euthanized. Eachrat eye is digitally imaged using ImagePro Plus at 4.0×. The surfacearea (pixels) and density (percent of area of interest) is quantitatedin the area directly beneath and on either side of the filter disk. Ninesurface area measurements are obtained per eye. A mean angiogenicsurface area is obtained for each eye.

One of skill in the art could readily modify the above protocol to testthe effect of agonists and/or antagonists of VEGF-2 (e.g., VEGF-2antibodies) on VEGF-2 modulated neovascularization. In one modification,the filter disks might be soaked with an equimolar amount of VEGF-2 andVEGF-2 antibody. Alternatively, one could treat the corneas directlyonly with VEGF-2 polypeptides and administer VEGF-2 antibodysystemically, via intraperitoneal, intravenous, or subcutaneousinjection. When systemic injection is used, for example, the rat may begiven one or more doses of between 0.1 to 10 mg/kg.

Example 24 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels

Experimental Design

The experimental protocol includes:

1. Diabetic db+/db+ Mouse Model.

To demonstrate that VEGF-2 accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA (1982)).Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice(db+/db+) have elevated blood glucose, increased or normal insulinlevels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Animals

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Surgical Wounding

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

VEGF-2 is administered using at a range different doses of VEGF-2, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Experimental Design

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) VEGF-2.

Measurement of Wound Area and Closure

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]Histology

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with VEGF-2. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Immunohistochemistry

Re-Epithelialization

Tissue sections are stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Cell Proliferation Marker

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Statistical Analysis

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

2. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that VEGF-2 can accelerate the healing process, theeffects of multiple topical applications of VEGF-2 on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone is assessed.

Animals

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

Surgical Wounding

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8 for Figure. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue was no longer visibleand the wound is covered by a continuous epithelium.

VEGF-2 is administered using at a range different doses of VEGF-2, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Experimental Design

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) VEGF-2 treated groups.

Measurement of Wound Area and Closure

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1was 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]Histology

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining wasperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin was improved by treatmentwith VEGF-2. A calibrated lens micrometer was used by a blinded observerto determine the distance of the wound gap.

Statistical Analysis

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Example 25 Specific Peptide Fragments to Generate VEGF-2 MonoclonalAntibodies

Four specific peptides (designated SP-40, SP-41, SP-42 and SP-43) havebeen generated. These will be used to generate monoclonal antibodies toanalyze VEGF-2 processing. The peptides are shown below:

1. “SP-40”: MTVLYPEYWKMY (amino acids 70-81 in SEQ ID NO: 18) 2.“SP-41”: KSIDNEWRKTQSMPREV (amino acids 120-136 (note C−>Smutation at position 131) in SEQ ID NO: 18) 3. “SP-42”: MSKLDVYRQVHSIIRR(amino acids 212-227 in SEQ ID NO: 18) 4. “SP-43”: MFSSDAGDDSTDGFHDI(amino acids 263-279 in SEQ ID NO: 18)

Example 26 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofVEGF-2 in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7-10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3-4 weeks.

Experimental Procedure

Prior to beginning surgery, blood sample was drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs were shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements were made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated.

The 2 proximal and 2 distal lymphatic vessels and distal blood supply ofthe popliteal node are then and ligated by suturing. The popliteal lymphnode, and any accompanying adipose tissue, is then removed by cuttingconnective tissues.

Care was taken to control any mild bleeding resulting from thisprocedure. After lymphatics were occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (A J Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals were checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak was then observed. To evaluate the intensity of thelymphedema, we measured the circumference and volumes of 2 designatedplaces on each paw before operation and daily for 7 days. The effectplasma proteins have on lymphedema and determined if protein analysis isa useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements:

Under brief gas anesthetic to prevent limb movement, a cloth tape isused to measure limb circumference. Measurements are done at the anklebone and dorsal paw by 2 different people then those 2 readings areaveraged. Readings are taken from both control and edematous limbs.

Volumetric Measurements:

On the day of surgery, animals are anesthetized with Pentobarbital andare tested prior to surgery. For daily volumetrics animals are underbrief halothane anesthetic (rapid immobilization and quick recovery),both legs are shaved and equally marked using waterproof marker on legs.Legs are first dipped in water, then dipped into instrument to eachmarked level then measured by Buxco edema software (Chen/Victor). Datais recorded by one person, while the other is dipping the limb to markedarea.

Blood-Plasma Protein Measurements:

Blood is drawn, spun, and serum separated prior to surgery and then atconclusion for total protein and Ca²⁺ comparison.

Limb Weight Comparison:

After drawing blood, the animal is prepared for tissue collection. Thelimbs were amputated using a quillitine, then both experimental andcontrol legs were cut at the ligature and weighed. A second weighing isdone as the tibio-cacaneal joint was disarticulated and the foot wasweighed.

Histological Preparations:

The transverse muscle located behind the knee (popliteal) area isdissected and arranged in a metal mold, filled with freezeGel, dippedinto cold methylbutane, placed into labeled sample bags at −80 EC untilsectioning. Upon sectioning, the muscle was observed under fluorescentmicroscopy for lymphatics. Other immuno/histological methods arecurrently being evaluated.

Example 27 Method of Treatment Using Gene Therapy for Production ofVEGF-2 Polypeptide In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) comprising VEGF-2 operably linked to a promoterinto an animal to increase the expression of VEGF-2. Such gene therapyand delivery techniques and methods are known in the art, see, forexample, WO 90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151,5,580,859; Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479, Chao,J et al. (1997) Pharmacol. Res. 35(6):517-522, Wolff, J. A. (1997)Neuromuscul. Disord. 7(5):314-318, Schwartz, B. et al. (1996) Gene Ther.3(5):405-411, Tsurumi, Y. et al. (1996) Circulation 94(12):3281-3290(incorporated herein by reference).

The VEGF-2 polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The VEGF-2 polynucleotideconstructs may also be delivered directly into arteries. The VEGF-2polynucleotide constructs can be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the VEGF-2 polynucleotide may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.(1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995)Biol. Cell 85(1):1-7) which can be prepared by methods well known tothose skilled in the art.

The VEGF-2 vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Unlike othergene therapies techniques, one major advantage of introducing nakednucleic acid sequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The VEGF-2 construct can be delivered to the interstitial space oftissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. They may be conveniently delivered by injectioninto the tissues comprising these cells. They are preferably deliveredto and expressed in persistent, non-dividing cells which aredifferentiated, although delivery and expression may be achieved innon-differentiated or less completely differentiated cells, such as, forexample, stem cells of blood or skin fibroblasts. Preferably, they aredelivered by direct injection into the artery.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues, or directly intoarteries. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked VEGF-2 constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected VEGF-2 polynucleotide construct inarteries in vivo is determined as follows. Suitable template DNA forproduction of mRNA coding for VEGF-2 is prepared in accordance with astandard recombinant DNA methodology. The template DNA, which may beeither circular or linear, is either used as naked DNA or complexed withliposomes. The arteries of rabbits are then injected with variousamounts of the template DNA.

Hindlimb ischemia in rabbits is surgically induced, as described inExample 18. Immediately following this, five different sites in theadductor (2 sites), medial large (2 sites), and semimembranous muscles(1 site) are injected directly with plasmid DNA encoding VEGF-2 using a3 ml syringe and 2-gauge needle advanced through a small skin incision.The skin is then closed using 4.0 nylon.

The ability to rescue hindlimb ischemia is determined by measuring thenumber of capillaries in light microscopic sections taken from thetreated hindlimbs, compared to ischemic hindlimbs from untreatedrabbits, measurement of calf blood pressure, and intraarterial Dopplerguidewire measurement of flow velocity (Takeshita et al., J. Clin.Invest. 93:662-670 (1994)). The results of the above experimentation inrabbits can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using VEGF-2 polynucleotide nakedDNA.

Example 28 Method of Treatment Using Gene Therapy Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing VEGF-2 polypeptides, onto a patient. Generally, fibroblastsare obtained from a subject by skin biopsy. The resulting tissue isplaced in tissue-culture medium and separated into small pieces. Smallchunks of the tissue are placed on a wet surface of a tissue cultureflask, approximately ten pieces are placed in each flask. The flask isturned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and thechunks of tissue remain fixed to the bottom of the flask and fresh media(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) isadded. The flasks are then incubated at 37 degree C. for approximatelyone week.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding VEGF-2 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted VEGF-2.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the VEGF-2 gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the VEGF-2 gene (the packagingcells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether VEGF-2protein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads

Example 29 Method of Treatment Using Gene Therapy HomologousRecombination

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous VEGF-2 sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous VEGF-2, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of VEGF-2 so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousVEGF-2 sequence. This results in the expression of VEGF-2 in the cell.Expression may be detected by immunological staining, or any othermethod known in the art. Fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in DMEM+10% fetal calf serum.Exponentially growing or early stationary phase fibroblasts aretrypsinized and rinsed from the plastic surface with nutrient medium. Analiquot of the cell suspension is removed for counting, and theremaining cells are subjected to centrifugation. The supernatant isaspirated and the pellet is resuspended in 5 ml of electroporationbuffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mMdextrose). The cells are recentrifuged, the supernatant aspirated, andthe cells resuspended in electroporation buffer containing 1 mg/mlacetylated bovine serum albumin. The final cell suspension containsapproximately 3×10⁶ cells/ml. Electroporation should be performedimmediately following resuspension.

Plasmid DNA is prepared according to standard techniques. To construct aplasmid for targeting to the VEGF-2 locus, plasmid pUC18 (MBI Fermentas,Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplifiedby PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end.Two VEGF-2 non-coding sequences are amplified via PCR: one VEGF-2non-coding sequence (VEGF-2 fragment 1) is amplified with a HindIII siteat the 5′ end and an Xba site at the 3′ end; the other VEGF-2 non-codingsequence (VEGF-2 fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′ end. The CMV promoter and VEGF-2fragments are digested with the appropriate enzymes (CMV promoter—XbaIand BamHI; VEGF-2 fragment 1—XbaI; VEGF-2 fragment 2—BamHI) and ligatedtogether. The resulting ligation product is digested with HindIII, andligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 EC. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 30 VEGF-2 Transgenic Animals

The VEGF-2 polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate transgenic animals. In a specific embodiment, techniquesdescribed herein or otherwise known in the art, are used to expresspolypeptides of the invention in humans, as part of a gene therapyprotocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol. Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of VEGF-2 polypeptides, studying conditions and/or disordersassociated with aberrant VEGF-2 expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 31 VEGF-2 Knock-Out Animals

Endogenous VEGF-2 gene expression can also be reduced by inactivating or“knocking out” the VEGF-2 gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe VEGF-2 polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of VEGF-2 polypeptides, studying conditions and/or disordersassociated with aberrant VEGF-2 expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 32 Identification and Cloning of VH and VL Domains

One method to identify and clone VH and VL domains from cell linesexpressing a particular antibody is to perform PCR with VH and VLspecific primers on cDNA made from the antibody expressing cell lines.Briefly, RNA is isolated from the cell lines and used as a template forRT-PCR designed to amplify the VH and VL domains of the antibodiesexpressed by the EBV cell lines. Cells may be lysed in the TRIzol®reagent (Life Technologies, Rockville, Md.) and extracted with one fifthvolume of chloroform. After addition of chloroform, the solution isallowed to incubate at room temperature for 10 minutes, and thecentrifuged at 14,000 rpm for 15 minutes at 4° C. in a tabletopcentrifuge. The supernatant is collected and RNA is precipitated usingan equal volume of isopropanol. Precipitated RNA is pelleted bycentrifuging at 14,000 rpm for 15 minutes at 4° C. in a tabletopcentrifuge. Following centrifugation, the supernatant is discarded andwashed with 75% ethanol. Following washing, the RNA is centrifuged againat 800 rpm for 5 minutes at 4° C. The supernatant is discarded and thepellet allowed to air dry. RNA is the dissolved in DEPC water and heatedto 60° C. for 10 minutes. Quantities of RNA can determined using opticaldensity measurements.

cDNA may be synthesized, according to methods well-known in the art,from 1.5-2.5 micrograms of RNA using reverse transciptase and randomhexamer primers. cDNA is then used as a template for PCR amplificationof VH and VL domains. Primers used to amplify VH and VL genes are shownin Table 5. Typically a PCR reaction makes use of a single 5′ primer anda single 3′ primer. Sometimes, when the amount of available RNA templateis limiting, or for greater efficiency, groups of 5′ and/or 3′ primersmay be used. For example, sometimes all five VH-5′ primers and all JH3′primers are used in a single PCR reaction. The PCR reaction is carriedout in a 50 microliter volume containing 1×PCR buffer, 2 mM of eachdNTP, 0.7 units of High Fidelity Taq polymerase, 5′ primer mix, 3′primer mix and 7.5 microliters of cDNA. The 5′ and 3′ primer mix of bothVH and VL can be made by pooling together 22 pmole and 28 pmole,respectively, of each of the individual primers. PCR conditions are: 96°C. for 5 minutes; followed by 25 cycles of 94° C. for 1 minute, 50° C.for 1 minute, and 72° C. for 1 minute; followed by an extension cycle of72° C. for 10 minutes. After the reaction is completed, sample tubes arestored 4° C.

TABLE 3 Primer Sequences Used to Amplify VH and VL domains. Primer nameSEQ ID NO Primer Sequence (5′-3′) VH Primers Hu VH1-5′ 36CAGGTGCAGCTGGTGCAGTCTGG Hu VH2-5′ 37 CAGGTCAACTTAAGGGAGTCTGG Hu VH3-5′38 GAGGTGCAGCTGGTGGAGTCTGG Hu VH4-5′ 39 CAGGTGCAGCTGCAGGAGTCGGGHu VH5-5′ 40 GAGGTGCAGCTGTTGCAGTCTGC Hu VH6-5′ 41CAGGTACAGCTGCAGCAGTCAGG Hu JH1,2-5′ 42 TGAGGAGACGGTGACCAGGGTGCCHu JH3-5′ 43 TGAAGAGACGGTGACCATTGTCCC Hu JH4,5-5′ 44TGAGGAGACGGTGACCAGGGTTCC Hu JH6-5′ 45 TGAGGAGACGGTGACCGTGGTCCCVL Primers Hu Vkappa1-5′ 46 GACATCCAGATGACCCAGTCTCC Hu Vkappa2a-5′ 47GATGTTGTGATGACTCAGTCTCC Hu Vkappa2b-5′ 48 GATATTGTGATGACTCAGTCTCCHu Vkappa3-5′ 49 GAAATTGTGTTGACGCAGTCTCC Hu Vkappa4-5′ 50GACATCGTGATGACCCAGTCTCC Hu Vkappa5-5′ 51 GAAACGACACTCACGCAGTCTCCHu Vkappa6-5′ 52 GAAATTGTGCTGACTCAGTCTCC Hu Vlambda1-5′ 53CAGTCTGTGTTGACGCAGCCGCC Hu Vlambda2-5′ 54 CAGTCTGCCCTGACTCAGCCTGCHu Vlambda3-5′ 55 TCCTATGTGCTGACTCAGCCACC Hu Vlambda3b-5′ 56TCTTCTGAGCTGACTCAGGACCC Hu Vlambda4-5′ 57 CACGTTATACTGACTCAACCGCCHu Vlambda5-5′ 58 CAGGCTGTGCTCACTCAGCCGTC Hu Vlambda6-5′ 59AATTTTATGCTGACTCAGCCCCA Hu Jkappa1-3′ 60 ACGTTTGATTTCCACCTTGGTCCCHu Jkappa2-3′ 61 ACGTTTGATCTCCAGCTTGGTCCC Hu Jkappa3-3′ 62ACGTTTGATATCCACTTTGGTCCC Hu Jkappa4-3′ 63 ACGTTTGATCTCCACCTTGGTCCCHu Jkappa5-3′ 64 ACGTTTAATCTCCAGTCGTGTCCC Hu Jlambda1-3′ 65CAGTCTGTGTTGACGCAGCCGCC Hu Jlambda2-3′ 66 CAGTCTGCCCTGACTCAGCCTGCHu Jlambda3-3′ 67 TCCTATGTGCTGACTCAGCCACC Hu Jlambda3b-3′ 68TCTTCTGAGCTGACTCAGGACCC Hu Jlambda4-3′ 69 CACGTTATACTGACTCAACCGCCHu Jlambda5-3′ 70 CAGGCTGTGCTCACTCAGCCGTC Hu Jlambda6-3′ 71AATTTTATGCTGACTCAGCCCCA

PCR samples are then electrophoresed on a 1.3% agarose gel. DNA bands ofthe expected sizes (˜506 base pairs for VH domains, and 344 base pairsfor VL domains) can be cut out of the gel and purified using methodswell known in the art. Purified PCR products can be ligated into a PCRcloning vector (TA vector from Invitrogen Inc., Carlsbad, Calif.).Individual cloned PCR products can be isolated after transfection of E.coli and blue/white color selection. Cloned PCR products may then besequenced using methods commonly known in the art.

The PCR bands containing the VH domain and the VL domains can also beused to create full-length Ig expression vectors. VH and VL domains canbe cloned into vectors containing the nucleotide sequences of a heavy(e.g., human IgG1 or human IgG4) or light chain (human kappa or humanlambda) constant regions such that a complete heavy or light chainmolecule could be expressed from these vectors when transfected into anappropriate host cell. Further, when cloned heavy and light chains areboth expressed in one cell line (from either one or two vectors), theycan assemble into a complete functional antibody molecule that issecreted into the cell culture medium. Methods using polynucleotidesencoding VH and VL antibody domains to generate expression vectors thatencode complete antibody molecules are well known within the art.

Example 33 BIAcore Analysis of the Affinity of VEGF-2 BindingPolypeptides

Binding of VEGF-2 antibodies to VEGF-2, for example, can be analyzed byBIAcore analysis. Either VEGF-2 (or other antigen to which one wants toknow the affinity of a VEGF-2 antibody) or VEGF-2 antibody can becovalently immobilized to a BIAcore sensor chip (CM5 chip) via aminegroups usingN-ethyl-N′-(dimethylaminopropyl)carboiimide/N-hydroxysuccinimidechemistry. Various dilutions of VEGF-2 antibodies or VEGF-2 (or otherantigen to which one wants to know the affinity of a VEGF-2 antibody),respectively, are flowed over the derivatized CM5 chip in flow cells at25 microliters/min for a total volume of 50 microliters. The amount ofbound protein is determined during washing of the flow cell with HBSbuffer (10 mM HEPES, pH7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactantp20). Binding specificity for the protein of interest is determined bycompetition with soluble competitor in the presence the protein ofinterest.

The flow cell surface can be regenerated by displacing bound protein bywashing with 20 microliters of 10 mM glycins-HCl, pH2.3. For kineticanalysis, the flow cells are tested at different flow rates anddifferent polypeptide densities on the CM5 chip. The on-rates andoff-rates can be determined using the kinetic evaluation program in aBIAevaluation 3 software.

BIAcore technology may also be utilized, for example, to quantitativelydetermine the ability of an anti-VEGF-2 antibody to inhibit the abilityof VEGF-2 to bind to its receptor.

Example 34 Endothelial Cell Proliferation Assay

VEGF-2 treatment induces vascular endothelial cells as well as lymphaticendothelial cells to undergo proliferation. One may use the followingassay to follow this activity in VEGF-2 treated cells. Additionally, onemay also use this assay to determine if VEGF-2 antibodies are able toinhibit the ability of VEGF-2 proteins to induce proliferation ofendothelial cells. The following protocol demonstrates the assay to testthe inhibitory activity of VEGF-2 antibodies, however, one of skill inthe art could readily modify this assay to omit the VEGF-2 antibodies inorder to test VEGF-2 proteins' ability to induce endothelial cellproliferation.

Continuous Subculture of Bovine Lymphatic Endothelial Cells (bLEC)

Bovine lymphatic endothelial cells (bLEC) cells (ATCC No. PTA-1149) aregrown in complete medium (DMEM, 10% of heat-inactivated FBS(Biowhittaker, Cat. #14-502F), 100 U/ml penicillin, 100 ug/mlstreptomycin, 2 mM glutamine, 5 ml/500 ml media of Non-Essential AminoAcids Solution (NEAA), 150 ug/ml bovine brain extract (bovine brainextract is prepared as described in Maciag et al., Proc. Natl. Acad.Sci. USA 76, 5674-5678 (1979)), 100 ug/ml heparin) in a 75 cm2 flask.When confluence is reached, the culture medium is removed from the 75cm2 flask, and cells are rinsed once with 20 ml of PBS without calciumand without magnesium, and then covered with 4 ml of trypsin-EDTA andreturned to the incubator for 3-5 minutes. The cells are then detachedfrom the plastic surface by gentle agitation, adding 4 ml of completemedium to inactivate the trypsin. The cell suspension is nexttransferred to the 15 ml centrifuge tube and centrifuged at 1000 rpm for5 minutes to pellet cells. The supernatant is removed and the cellpellet resuspended in complete medium. The cells are split 1:3 andmaintained in CO₂ incubator at 37 degrees C. The medium is changed twiceweekly regardless of the need to split the cells.

VEGF-2 Proliferation Assay Protocol

Day 1: Plate bLECs at 3500 cells per well in a 96 well plate excludingouter rows and culture overnight in complete medium. The following day,

Day 2: Remove complete medium and add 100 microliters of starvationmedium: EBM (Clonetics Cat. #CC-3121), 0.5% FBS.

Day 3: Dilute VEGF-2 protein in starvation medium yielding a 3×concentration. In 12 well plates, make serial dilutions of each antibody(using the 3× concentration of VEGF-2 in starvation medium as thediluent) for a total of 14 dilutions. The final concentrations afteradding the antibody to the experimental plates should range from 4000ng/ml to 0.0006 ng/ml (3-fold dilutions) in triplicates. Transfer 50microliters of each dilution starting from the least amount of antibodyto the 96 well plate. Incubate the plates for 3 days at 37 degreesCelsius, 5% CO₂.

Day 6: Supplement each well with 50 microliters of starvation mediumcontaining 3H-thymidine 0.5 mCi per well (6.7 Ci/mM) and incubate cellsfor an additional 18-20 hours.

Day 7: Freeze plates at −80 degrees C. After 2-3 h, thaw the plates.Harvest cells and measure 3H-thymidine incorporation.

Example 35 Elk-1 Phosphorylation Assay

VEGF-2 induces a signalling cascade in VEGF-2 responsive cells thatactivates a kinase which phosphorylates Elk-1 protein. One may use thefollowing assay to follow this activity in VEGF-2 treated cells.Additionally, one may also use this assay to determine if VEGF-2antibodies are able to inhibit the ability of VEGF-2 proteins to induceElk-1 phosphorylation. The following protocol demonstrates the assay totest the inhibitory activity of VEGF-2 antibodies, however, one of skillin the art could readily modify this assay to omit the VEGF-2 antibodiesin order to test VEGF-2 proteins' ability to induce Elk-1phosphorylation.

Briefly, 96-well flat bottom tissue culture plates are seeded withbovine lymphatic endothelial cells (bLEC) at 25,000 cells per well in a100 microliter volume of complete growth medium (EGM-MV from CloneticsCorporation, Cat. No. CC-4143) and incubated overnight at 37 C in 5%CO₂. Working stock solutions of VEGF-2 (e.g., full length protein or thesecreted form of VEGF-2) at 0.2 microgram/milliliter in PBS+0.05% BSA(low endotoxin) are prepared.

In a separate assay plate, mix either 6 ng of full length or 2 ng of thesecreted form of VEGF-2 protein with 1,500 ng (which represents 100×molar excess of antibody) of VEGF-2 antibody, and adjust the totalvolume to 100 microliters with Human Endothelial-Serum-Free Medium (LifeTechnologies, Cat. No. 11111-044) (SFM). The final concentration fulllength VEGF-2, secreted VEGF-2 and antibody are 60 ng/mL, 20 ng/mL and15 ug/mL, respectively). If the antibody samples are conditioned media,use the total IgG concentrations to calculate the amount of antibodyneeded in the assay. VEGF-2-antibody complexes are allowed to form for 1hour at room temperature.

While the antibody-antigen complexes are forming, remove the completegrowth medium from the cells and replace it with SFM starvation medium,and incubate the cells for 5 Incubate for one hour at 37 C in 5% CO₂.After the one hour incubation of the cells is over, decant thestarvation media from the cells and transfer 50 microliters of eachVEGF-2/VEGF-2 antibody sample from the assay plate to the cell plate.Then, incubate the cell plate for 15 minutes at 37 C. Followingincubation, decant the liquid containing VEGF-2-antiVEGF-2 complexesfrom the cells and 50 microliters per well of ice-cold lysis buffer (20mM Tris-Cl (pH 7.5), 250 mM NaCl, 0.5% NP-40, 10% glycerol, 3 mM EDTA, 3mM EGTA, 0.1 mM sodium orthovanadate, 1 mM NaF, 0.5 mM DTT [add fresh],1× Roche Complete protease inhibitor [add fresh]) and let stand 1-3minutes. The lysate should be used immediately in kinase assay or storedat −70 C.

Kinase Assay:

Dilute GST-Elk1 fusion protein (Cell Signaling Technologies #9184, orBoston Biologicals #1010) in PBS to 10 ug/mL, and add to a 96-well DynexMicrolite 2 plate (Catalogue #7417) at 50 microliters per well. Tap theplate gently to get liquid to cover the bottom completely. Incubateovernight at room temperature or for 1 hour at 37 C. Wash the plate oncewith 250 microliters per well of wash buffer (0.05% Tween 20, PBS+PBST).Next, block unoccupied binding sites in the wells with blocking buffer(1.0% Nonfat Dry Milk, PBST) at 150 microliters per well and incubatefor 1 hour at room temperature. The plate are then washed three timeswith 250 microliters per well of wash buffer. To each well in the assayplate, and 15 microliters of samples (in duplicates) and 10 microlitersof water. Initiate kinase reaction by adding 25 microliters per well of2× kinase buffer (2× Kinase Reaction Buffer (100 mM Hepes (pH 7.5), 20mM MgCl2, 5 mM NaF, 0.2 mM sodium orthovanadate, 1 mM DTT [add fresh], 1mM ATP [add fresh]) to each well. Include purified, activated andunactivated ERK1/2 kinase (Stratagene, #206110 and #206120,respectively) as controls. Incubate at room temperature for 1 hour (thereaction is linear between 1 to 3 hours).

Following incubation of the lysate or kinase controls with GST-Elk-1fusion protein, wash the plate three times with 250 microliters per wellof wash buffer. Then, dilute anti-phospho-Elk1 antibody 1:1000 (CellSignaling Technologies, #9181) with antibody diluent (0.1% BSA, PBST),and add 50 microliters to each well. Incubate at room temperature for 1hour.

Then, wash the plate three times with 250 microliters per well of washbuffer. Dilute Zymax goat anti-rabbit IgG-alkaline phosphatase (ZymedLaboratories Inc., #81-6122) 1:4000 with antibody diluent. Transfer 50microliters per well of diluted antibody to each well, and incubate for1 hour at RT. Wash three times with 250 microliters per well of washbuffer. Then add 50 microliters per well BM chemiluminescent ELISA APsubstrate (Roche Molecular Biochemicals, #1759779) prepared according tothe “ELISA directions” in the package. Incubate at room temperature for12 minutes before reading in a luminometer.

Example 36 Dorsal Chamber Model to Study Effect of VEGF-2 Antibodies ofTumor Vascularization

Characterization of the multiple aspects of microvascular physiology intransparent window systems in mice have provided valuable data onangiogenesis, inflammation, microvascular transport, tissue rejection,and tumor physiology (Melder, R. J et al., Biophys. J 69: 2131-2138,(1995); Fukumura, D. et al Cancer Res. 55: 4824-4829, (1995); Yamada, S.et al., Blood, 86: 3487-3492, (1995); Yamada, S., et al., Blood, 86:4707-4708, (1995); Melder, R. J., et al., Microvas. Res. 50, 35-44,(1995); Melder, R. J. et al., Nature Medicine 2:992-997, (1996);Dellian, M., et al., Am. J. Path. 149: 59-71, (1996); and Leunig M., etal., Cancer Res 52: 6553-60 (1992)). This assay may be used to test thehypothesis that VEGF-2 polypeptides administered directly to theinterstitial compartment of the skin or pial surface will induce achange in the structure and function of the microvasculature. Thesestudies will specifically characterize activities on existingvasculature within the observation window and neogenic vasculaturedeveloping in response to these proteins in implanted gels. Thefollowing observations will be made:

a) length, diameter and density of the existing vascular network in theskin or pial surface in response to treatment with VEGF-2 polypeptides;

b) blood flow velocity and leukocyte flux in the treated vascular bed;

c) the frequency of rolling and adherent leukocytes in the treatedvascular bed;

d) the permeability of existing vascular network in the skin or pialsurface in response to treatment with VEGF-2 polypeptides;

e) the angiogenic response to VEGF-2 polypeptides in implanted collagendisks within the window preparations;

f) blood flow velocity leukocyte flux and frequency of rolling andadherent leukocytes in the implanted collagen disks within the windowpreparations;

g) The permeability of angiogenic vascular networks in response toVEGF-2 polypeptides in implanted collagen disks within the skin orcranial window preparations.

For this assay, Swiss nu/nu mice are used. The advantages of using Swissnu/nu mice are several-fold, including a) reducing the possibility ofimmune response over the period of study, b) improving the optics of thesystem due to lack of pigmentation and hair in the skin, c) maintaininghistorical consistency with similar previous studies.

Each experimental and control group will have seven mice. Male mice arepreferred since they are larger and will have more skin for surgicalwindow implants. Samples for testing will include VEGF-2 polypeptidesand recombinant protein controls. Each study examining proteinactivities on existing vascular beds will consist of five experimentalgroups:

Group 1: Test sample (dose 1) in buffer

Group 2: Test sample (dose 2) in buffer

Group 3: Test sample (dose 3) in buffer

Group 4: Buffer and BSA control

Group 5: Positive Control (bFGF, 10 ng)

Sterile protein solutions will be administered as a 10 μl volumedirectly into the window preparation of mice with dorsal skin windows.Alternatively, sterile collagen/sucralfate disks containing proteinconcentrations as listed above will be placed into the windowpreparations for evaluation of the angiogenic potential. Sterileantibody solutions will be administered intravenously.

These experiments are designed to test the hypothesis that VEGF-2polypeptides administered to the extravascular compartment of the skinwill induce a change in the structure and function of the existingcapillaries and postcapillary venules. Administration of polypeptides incollagen disks will examine their potential for modulating angiogenesis.

Methods

Animal Preparation.

The surgical procedures are performed in Swiss nude mice. For thesurgical procedures, animals (20-30 g) are anesthetized with asub-cutaneous injection of a cocktail of 90 mg Ketamine and 9 mgXylazine per kg body weight. All surgical procedures are performed underaseptic conditions in a horizontal laminar flow hood, with all equipmentbeing steam, gas, or chemically sterilized. Sterility of the bench aremaintained by U.V. lights when not in use. During surgery, the bodytemperature of the animals is kept constant by means of a heated worksurface. All mice are housed individually in microisolator cages and allmanipulations are done in laminar flow hoods. Following surgery, animalsare observed for any discomfort/distress. The criteria for discomfortinclude, but are not limited to: loss of body weight (20%), inability toambulate, evidence of self-mutilation, or inability to eat or drink.Buprenorphine (0.1 mg/kg q 12 h) is used as an analgesic for 3 days postimplantation. Any animal exhibiting the signs of discomfort for 3 dayspost surgery, are euthanized with CO₂ inhalation.

Dorsal Skin Chamber Implantation:

Chambers are implanted as described in Leunig et al., Cancer Res52:6553-6560 (1992). Briefly, the chamber is positioned such that thechamber is positioned over a double layer of skin (i.e., a “pinch ofskin”) that extends above the dorsal surface. The full thickness of oneside of the dorsal skin flap is removed in a circular area 15 mm indiameter. The second side of the flap (consisting of epidermis, fascia,and striated muscle) is positioned on the frame of the chamber and theopening (“window”) is covered with a sterile, glass coverslip. Thechamber is held in place with suture (silk, 4-0) which is threadedthrough the extended skin and holes along the top of the chamber. Miceare allowed to recover 72 hours.

Following this recovery, each mouse is positioned in a transparent,polycarbonate tube (25 mm diameter) for treatment. The coverslips arecarefully removed, followed by addition of treatment factor(s). Afteraddition of treatment factors, a new, sterile coverslip will then placedon the viewing surface. Measurements are made by morphometric analysisusing a CCD and SIT camera, S-VHS videocasette recorder and directdigital image acquisition. Mice with implanted chambers are observed for28 days, as indicated in the flow charts.

Measurements.

Mice are anesthetized with s.c. injection of a cocktail of 90 mgKetamine and 9 mg Xylazine per kg body weight, then positioned on asterile plastic stage assembly. Vascular maps of the window will then bemade using transillumination (dorsal skin window) or following aninjection of 100 μl of BSA-FITC (1 mg/ml, i.v.) and epi-illumination(cranial window). Video recordings of vascular beds are made at a rangeof magnifications (from 1× to 40×) as well as digital frames foroff-line analysis. Images are quantified for vascular density, bloodflow velocity and vascular dimensions (for shear rate analysis). Inaddition, circulating leukocyte interactions are evaluated by injectionwith 10 μl of Rhodamine 6-G followed by video microscopy of thecapillaries and postcapillary venules. Permeability measurements aremade from off-line analysis of images of BSA transport at 5, 10, 15 and20 min. following BSA-FITC injection. Capillary density determinationsof normal and angiogenic vascular beds are made from offline analysis ofvideo tapes. Five sets of observations of experimental and control miceare at seven day intervals (total of 28 days).

Example 37 Colon Carcinoma LS174 T Dorsal Chamber Model

The colon carcinoma, LS174T, produces/secretes VEGF-2 polypeptide. It istherefore particularly interesting to test whether treatment with VEGF-2antibodies slows or arrests LS174T tumor growth, or even effects LS174Ttumor regression. To test this hypothesis the dorsal chamber modeldescribed above, may be adapted to study tumor growth andvascularization within the dorsal chamber.

Three days after a dorsal chamber is implanted, 2 microliters ofpelleted LS174T tumor cell suspension (containing approximately 2×10⁵cells) is innoculated onto the striated muscle layer of the subcutaneoustissue in the chamber. The innoculated tumor is then left for a periodof time to allow it to “take” and grow to a specified size (e.g., 4-6 mmin diameter) prior to beginning VEGF-2 treatment.

Mice are initially injected with 0.4 milligrams of VEGF-2 antibody,followed by injections of 0.2 milligrams of VEGF-2 antibody given atfive day intervals for 30 days. Injections may be givenintraperitoneally (i.p.) or intravenously (i.v.).

Parameters that may be measured to assess the effect of VEGF-2 treatmenton tumor growth include tumor size and (endothelial and lymphatic)vascular density.

Additionally, this assay may easily be modified to test the effect ofVEGF-2 treatment on other tumors, regardless of their production ofendogenous VEGF-2 polypeptide.

Example 38 Effect of VEGF-2 Antibody Treatment on MDA-MB-231 Tumor

The MDA-MB-231 cell line (ATCC # HTB-26) is a breast cancer cell line.The following assay may be used to test whether treatment with VEGF-2antibodies slows or arrests MDA-MB-231 tumor growth, or effectsMDA-MB-231 tumor regression. While the following example outlines anexperimental protocol involving MDA-MB-231 cells, one of skill in theart could easily modify this protocol to test the effect of treatmentwith VEGF-2 antibodies on other tumor types.

On day zero, mice are injected in the mammary pads with one millionMDA-MB-231 cells. The implanted tumor is allowed to grow to a 2 mm×2 mmwhich usually takes about 5 days. After the tumor has reached the 2×2mm2 size, an animal is given an initial dose Of 04. Milligrams of VEGF-2antibody. Thereafter, 0.2 milligrams of VEGF-2 antibody is administeredon the fifth and 10th days after the initial injection. Additionally,animals in certain experimental groups may also be treated with 5 mg/kgof Taxol (or other suitable dose of another chemotherapeutic agent)daily.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. A method of treating or ameliorating cancer or tumor mediated byhuman VEGF-2, comprising administering to a human an isolated antibodyor antigen binding fragment thereof that specifically binds to a humanVEGF-2 polypeptide, said antibody comprising: (i) an amino acid sequencethat is at least 96% identical to the amino acid sequence of the VHdomain of the antibody expressed by the hybridoma cell line depositedunder ATCC Deposit No. PTA-4095, wherein the amino acid sequencecomprises the amino acid sequences of the VHCDR1, VHCDR2, and VHCDR3regions of the antibody expressed by the hybridoma deposited under ATCCDeposit No. PTA-4095; and (ii) an amino acid sequence that is at least96% identical to the amino acid sequence of the VL domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VLCDR1, VLCDR2, and VLCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095.
 2. The method of claim 1, wherein the cancer or tumor is ofthe breast, brain, prostate or colon, a lymphangioma or Kaposi'ssarcoma.
 3. The method of claim 1, wherein the isolated antibody orfragment thereof is administered in combination with a chemotherapeuticagent.
 4. The method of claim 1, wherein the isolated antibody orfragment thereof is administered in combination with radiation therapy.5. The method of claim 1, wherein the isolated antibody or fragmentthereof is administered in combination with an anti-angiogenic agent. 6.The method of claim 1, wherein the isolated antibody or fragment thereofis administered at a dosage of 0.1 mg/kg to 100 mg/kg of the human'sbody weight.
 7. The method of claim 6, wherein the isolated antibody orfragment thereof is administered at a dosage of 0.1 mg/kg to 20 mg/kg ofthe human's body weight.
 8. The method of claim 7, wherein the isolatedantibody or fragment thereof is administered at a dosage of 1 mg/kg to10 mg/kg of the human's body weight.
 9. The method of claim 1, whereinthe isolated antibody or fragment thereof is administered intravenously.10. The method of claim 1, wherein the isolated antibody or fragmentthereof is administered in a pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient.
 11. The method ofclaim 1, wherein the isolated antibody is a monoclonal antibody.
 12. Themethod of claim 1, wherein the isolated antibody is a human antibody orhumanized antibody.
 13. The method of claim 1, wherein the antigenbinding antibody fragment is selected from the group consisting of Fab,Fab′, F(ab′)₂, Fv, single-chain Fv and disulfide-linked Fv.
 14. Themethod of claim 1, wherein the isolated antibody or fragment thereofbinds to a VEGF-2 polypeptide selected from the group consisting of: (a)a VEGF-2 polypeptide comprising amino acids 1-419 of SEQ ID NO:18; (b) aVEGF-2 polypeptide comprising amino acids 32-419 of SEQ ID NO:18; (c) aVEGF-2 polypeptide comprising amino acids 103-227 of SEQ ID NO:18; (d) aVEGF-2 polypeptide comprising amino acids 112-227 of SEQ ID NO:18; (e) adimeric VEGF-2 polypeptide consisting of two polypeptides eachconsisting of amino acids 103-227 of SEQ ID NO:18; (f) a dimeric VEGF-2polypeptide consisting of two polypeptides each consisting of aminoacids 112-227 of SEQ ID NO:18; (g) the amino acid sequence of thefull-length VEGF-2 polypeptide encoded by cDNA contained in ATCC DepositNumber 97149; (h) the amino acid sequence of the pro-protein VEGF-2polypeptide encoded by cDNA contained in ATCC Deposit Number 97149; (i)the amino acid sequence of the secreted VEGF-2 polypeptide encoded bycDNA contained in ATCC Deposit Number 97149; (j) the amino acid sequenceof the full-length VEGF-2 polypeptide encoded by cDNA contained in. ATCCDeposit Number 75698; (k) the amino acid sequence of the pro-proteinVEGF-2 polypeptide encoded by cDNA contained in ATCC Deposit Number75698; (l) the amino acid sequence of the secreted VEGF-2 polypeptideencoded by cDNA contained in ATCC Deposit Number 75698; and (m) theamino acid sequence of the secreted form of the VEGF-2 polypeptide ofSEQ ID NO:18.
 15. The method of claim 1, wherein the isolated antibodyor fragment thereof comprises: (i) an amino acid sequence that is atleast 97% identical to the amino acid sequence of the VH domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VHCDR1, VHCDR2, and VHCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095; (ii) an amino acid sequence that is at least 97% identical tothe amino acid sequence of the VL domain of the antibody expressed bythe hybridoma cell line deposited under ATCC Deposit No. PTA-4095,wherein the amino acid sequence comprises the amino acid sequences ofthe VLCDR1, VLCDR2, and VLCDR3 regions of the antibody expressed by thehybridoma deposited under ATCC Deposit No. PTA-4095; or (iii) an aminoacid sequence that is at least 97% identical to the amino acid sequenceof the VH domain of the antibody expressed by the hybridoma cell linedeposited under ATCC Deposit No. PTA-4095, wherein the amino acidsequence comprises the amino acid sequences of the VHCDR1, VHCDR2, andVHCDR3 regions of the antibody expressed by the hybridoma depositedunder ATCC Deposit No. PTA-4095; and an amino acid sequence that is atleast 97% identical to the amino acid sequence of the VL domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VLCDR1, VLCDR2, and VLCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095.
 16. The method of claim 15, wherein the isolated antibody orfragment thereof comprises: (i) an amino acid sequence that is at least98% identical to the amino acid sequence of the VH domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VHCDR1, VHCDR2, and VHCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095; (ii) an amino acid sequence that is at least 98% identical tothe amino acid sequence of the VL domain of the antibody expressed bythe hybridoma cell line deposited under ATCC Deposit No. PTA-4095,wherein the amino acid sequence comprises the amino acid sequences ofthe VLCDR1, VLCDR2, and VLCDR3 regions of the antibody expressed by thehybridoma deposited under ATCC Deposit No. PTA-4095; or (iii) an aminoacid sequence that is at least 98% identical to the amino acid sequenceof the VH domain of the antibody expressed by the hybridoma cell linedeposited under ATCC Deposit No. PTA-4095, wherein the amino acidsequence comprises the amino acid sequences of the VHCDR1, VHCDR2, andVHCDR3 regions of the antibody expressed by the hybridoma depositedunder ATCC Deposit No. PTA-4095; and an amino acid sequence that is atleast 98% identical to the amino acid sequence of the VL domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VLCDR1, VLCDR2, and VLCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095.
 17. The method of claim 16, wherein the isolated antibody orfragment thereof comprises: (i) an amino acid sequence that is at least99% identical to the amino acid sequence of the VH domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VHCDR1, VHCDR2, and VHCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095; (ii) an amino acid sequence that is at least 99% identical tothe amino acid sequence of the VL domain of the antibody expressed bythe hybridoma cell line deposited under ATCC Deposit No. PTA-4095,wherein the amino acid sequence comprises the amino acid sequences ofthe VLCDR1, VLCDR2, and VLCDR3 regions of the antibody expressed by thehybridoma deposited under ATCC Deposit No. PTA-4095; or (iii) an aminoacid sequence that is at least 99% identical to the amino acid sequenceof the VH domain of the antibody expressed by the hybridoma cell linedeposited under ATCC Deposit No. PTA-4095, wherein the amino acidsequence comprises the amino acid sequences of the VHCDR1, VHCDR2, andVHCDR3 regions of the antibody expressed by the hybridoma depositedunder ATCC Deposit No. PTA-4095; and an amino acid sequence that is atleast 99% identical to the amino acid sequence of the VL domain of theantibody expressed by the hybridoma cell line deposited under ATCCDeposit No. PTA-4095, wherein the amino acid sequence comprises theamino acid sequences of the VLCDR1, VLCDR2, and VLCDR3 regions of theantibody expressed by the hybridoma deposited under ATCC Deposit No.PTA-4095.
 18. The method of claim 17, wherein the isolated antibody orfragment thereof comprises: (i) the amino acid sequence of the VH domainof the antibody expressed by the hybridoma cell line deposited underATCC Deposit No. PTA-4095; (ii) the amino acid sequence of the VL domainof the antibody expressed by the hybridoma cell line deposited underATCC Deposit No. PTA-4095; or (iii) the amino acid sequence of the VHdomain of the antibody expressed by the hybridoma cell line depositedunder ATCC Deposit No. PTA-4095; and the amino acid sequence of the VLdomain of the antibody expressed by the hybridoma cell line depositedunder ATCC Deposit No. PTA-4095.
 19. A method of treating orameliorating cancer or tumor mediated by human VEGF-2, comprisingadministering to a human an isolated antibody or antigen bindingfragment thereof that specifically binds to a human VEGF-2 polypeptide,said antibody comprising: (i) an amino acid sequence that is at least96% identical to the amino acid sequence of the VH domain of SEQ ID NO:79, wherein the amino acid sequence comprises: a VHCDR1 consisting ofthe amino acid sequence of amino acid residues 26-35 of SEQ ID NO: 79; aVHCDR2 consisting of the amino acid sequence of amino acid residues50-66 of SEQ ID NO: 79; and a VHCDR3 consisting of the amino acidsequence of amino acid residues 99-107 of SEQ ID NO: 79; and (ii) anamino acid sequence that is at least 96% identical to the amino acidsequence of the VL domain of SEQ ID NO: 79, wherein the amino acidsequence comprises: a VLCDR1 consisting of the amino acid sequence ofamino acid residues 158-170 of SEQ ID NO: 79; a VLCDR2 consisting of theamino acid sequence of amino acid residues 186-192 of SEQ ID NO: 79; anda VLCDR3 consisting of the amino acid sequence of amino acid residues225-236 of SEQ ID NO:
 79. 20. The method of claim 19, wherein the canceror tumor is of the breast, brain, prostate or colon, a lymphangioma orKaposi's sarcoma.
 21. The method of claim 19, wherein the isolatedantibody or fragment thereof is administered in combination with achemotherapeutic agent.
 22. The method of claim 19, wherein the isolatedantibody or fragment thereof is administered in combination withradiation therapy.
 23. The method of claim 19, wherein the isolatedantibody or fragment thereof is administered in combination with ananti-angiogenic agent.
 24. The method of claim 19, wherein the isolatedantibody or fragment thereof is administered at a dosage of 0.1 mg/kg to100 mg/kg of the human's body weight.
 25. The method of claim 24,wherein the isolated antibody or fragment thereof is administered at adosage of 0.1 mg/kg to 20 mg/kg of the human's body weight.
 26. Themethod of claim 25, wherein the isolated antibody or fragment thereof isadministered at a dosage of 1 mg/kg to 10 mg/kg of the human's bodyweight.
 27. The method of claim 19, wherein the isolated antibody orfragment thereof is administered intravenously.
 28. The method of claim19, wherein the isolated antibody or fragment thereof is administered ina pharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient.
 29. The method of claim 19, wherein the isolatedantibody is a monoclonal antibody.
 30. The method of claim 19, whereinthe isolated antibody is a human antibody or humanized antibody.
 31. Themethod of claim 19, wherein the antigen binding fragment is selectedfrom the group consisting of Fab, Fab′, F(ab′)₂, Fv, single-chain Fv anddisulfide-linked Fv.
 32. The method of claim 19, wherein the isolatedantibody or fragment thereof binds to a VEGF-2 polypeptide selected fromthe group consisting of: (a) a VEGF-2 polypeptide comprising amino acids1-419 of SEQ ID NO:18; (b) a VEGF-2 polypeptide comprising amino acids32-419 of SEQ ID NO:18; (c) a VEGF-2 polypeptide comprising amino acids103-227 of SEQ ID NO:18; (d) a VEGF-2 polypeptide comprising amino acids112-227 of SEQ ID NO:18; (e) a dimeric VEGF-2 polypeptide consisting oftwo polypeptides each consisting of amino acids 103-227 of SEQ ID NO:18;(f) a dimeric VEGF-2 polypeptide consisting of two polypeptides eachconsisting of amino acids 112-227 of SEQ ID NO:18; (g) the amino acidsequence of the full-length VEGF-2 polypeptide encoded by cDNA containedin ATCC Deposit Number 97149; (h) the amino acid sequence of thepro-protein VEGF-2 polypeptide encoded by cDNA contained in ATCC DepositNumber 97149; (i) the amino acid sequence of the secreted VEGF-2polypeptide encoded by cDNA contained in ATCC Deposit Number 97149; (j)the amino acid sequence of the full-length VEGF-2 polypeptide encoded bycDNA contained in ATCC Deposit Number 75698; (k) the amino acid sequenceof the pro-protein VEGF-2 polypeptide encoded by cDNA contained in ATCCDeposit Number 75698; (l) the amino acid sequence of the secreted VEGF-2polypeptide encoded by cDNA contained in ATCC Deposit Number 75698; and(m) the amino acid sequence of the secreted form of the VEGF-2polypeptide of SEQ ID NO:18.
 33. The method of claim 19, wherein theisolated antibody or fragment thereof comprises: (i) an amino acidsequence that is at least 97% identical to the amino acid sequence ofthe VH domain of SEQ ID NO: 79, wherein the amino acid sequencecomprises: a VHCDR1 consisting of the amino acid sequence of amino acidresidues 26-35 of SEQ ID NO: 79; a VHCDR2 consisting of the amino acidsequence of amino acid residues 50-66 of SEQ ID NO: 79; and a VHCDR3consisting of the amino acid sequence of amino acid residues 99-107 ofSEQ ID NO: 79; (ii) an amino acid sequence that is at least 97%identical to the amino acid sequence of the VL domain of SEQ ID NO: 79,wherein the amino acid sequence comprises: a VLCDR1 consisting of theamino acid sequence of amino acid residues 158-170 of SEQ ID NO: 79; aVLCDR2 consisting of the amino acid sequence of amino acid residues186-192 of SEQ ID NO: 79; and a VLCDR3 consisting of the amino acidsequence of amino acid residues 225-236 of SEQ ID NO: 79; or (iii) anamino acid sequence that is at least 97% identical to the amino acidsequence of the VH domain of SEQ ID NO: 79, wherein the amino acidsequence comprises: a VHCDR1 consisting of the amino acid sequence ofamino acid residues 26-35 of SEQ ID NO: 79; a VHCDR2 consisting of theamino acid sequence of amino acid residues 50-66 of SEQ ID NO: 79; and aVHCDR3 consisting of the amino acid sequence of amino acid residues99-107 of SEQ ID NO: 79; and an amino acid sequence that is at least 97%identical to the amino acid sequence of the VL domain of SEQ ID NO: 79,wherein the amino acid sequence comprises: a VLCDR1 consisting of theamino acid sequence of amino acid residues 158-170 of SEQ ID NO: 79; aVLCDR2 consisting of the amino acid sequence of amino acid residues186-192 of SEQ ID NO: 79; and a VLCDR3 consisting of the amino acidsequence of amino acid residues 225-236 of SEQ ID NO:
 79. 34. The methodof claim 33, wherein the isolated antibody or fragment thereofcomprises: (i) an amino acid sequence that is at least 98% identical tothe amino acid sequence of the VH domain of SEQ ID NO: 79, wherein theamino acid sequence comprises: a VHCDR1 consisting of the amino acidsequence of amino acid residues 26-35 of SEQ ID NO: 79; a VHCDR2consisting of the amino acid sequence of amino acid residues 50-66 ofSEQ ID NO: 79; and a VHCDR3 consisting of the amino acid sequence ofamino acid residues 99-107 of SEQ ID NO: 79; (ii) an amino acid sequencethat is at least 98% identical to the amino acid sequence of the VLdomain of SEQ ID NO: 79, wherein the amino acid sequence comprises: aVLCDR1 consisting of the amino acid sequence of amino acid residues158-170 of SEQ ID NO: 79; a VLCDR2 consisting of the amino acid sequenceof amino acid residues 186-192 of SEQ ID NO: 79; and a VLCDR3 consistingof the amino acid sequence of amino acid residues 225-236 of SEQ ID NO:79; or (iii) an amino acid sequence that is at least 98% identical tothe amino acid sequence of the VH domain of SEQ ID NO: 79, wherein theamino acid sequence comprises: a VHCDR1 consisting of the amino acidsequence of amino acid residues 26-35 of SEQ ID NO: 79; a VHCDR2consisting of the amino acid sequence of amino acid residues 50-66 ofSEQ ID NO: 79; and a VHCDR3 consisting of the amino acid sequence ofamino acid residues 99-107 of SEQ ID NO: 79; and an amino acid sequencethat is at least 98% identical to the amino acid sequence of the VLdomain of SEQ ID NO: 79, wherein the amino acid sequence comprises: aVLCDR1 consisting of the amino acid sequence of amino acid residues158-170 of SEQ ID NO: 79; a VLCDR2 consisting of the amino acid sequenceof amino acid residues 186-192 of SEQ ID NO: 79; and a VLCDR3 consistingof the amino acid sequence of amino acid residues 225-236 of SEQ ID NO:79.
 35. The method of claim 34, wherein the isolated antibody orfragment thereof comprises: (i) an amino acid sequence that is at least99% identical to the amino acid sequence of the VH domain of SEQ ID NO:79, wherein the amino acid sequence comprises: a VHCDR1 consisting ofthe amino acid sequence of amino acid residues 26-35 of SEQ ID NO: 79; aVHCDR2 consisting of the amino acid sequence of amino acid residues50-66 of SEQ ID NO: 79; and a VHCDR3 consisting of the amino acidsequence of amino acid residues 99-107 of SEQ ID NO: 79; (ii) an aminoacid sequence that is at least 99% identical to the amino acid sequenceof the VL domain of SEQ ID NO: 79, wherein the amino acid sequencecomprises: a VLCDR1 consisting of the amino acid sequence of amino acidresidues 158-170 of SEQ ID NO: 79; a VLCDR2 consisting of the amino acidsequence of amino acid residues 186-192 of SEQ ID NO: 79; and a VLCDR3consisting of the amino acid sequence of amino acid residues 225-236 ofSEQ ID NO: 79; or (iii) an amino acid sequence that is at least 99%identical to the amino acid sequence of the VH domain of SEQ ID NO: 79,wherein the amino acid sequence comprises: a VHCDR1 consisting of theamino acid sequence of amino acid residues 26-35 of SEQ ID NO: 79; aVHCDR2 consisting of the amino acid sequence of amino acid residues50-66 of SEQ 1D NO: 79; and a VHCDR3 consisting of the amino acidsequence of amino acid residues 99-107 of SEQ ID NO: 79; and an aminoacid sequence that is at least 99% identical to the amino acid sequenceof the VL domain of SEQ ID NO: 79, wherein the amino acid sequencecomprises: a VLCDR1 consisting of the amino acid sequence of amino acidresidues 158-170 of SEQ ID NO: 79; a VLCDR2 consisting of the amino acidsequence of amino acid residues 186-192 of SEQ ID NO: 79; and a VLCDR3consisting of the amino acid sequence of amino acid residues 225-236 ofSEQ ID NO:
 79. 36. The method of claim 35, wherein the isolated antibodyor fragment thereof comprises: (i) the amino acid sequence of the VHdomain of SEQ ID NO: 79; (ii) the amino acid sequence of the VL domainof SEQ ID NO: 79; or (iii) the amino acid sequence of the VH domain ofSEQ ID NO: 79; and the amino acid sequence of the VL domain of SEQ IDNO: 79.